Substituted quinobenzoxazine analogs

ABSTRACT

The present invention relates to quinobenzoxazines analogs having the general formula:  
                 
and pharmaceutically acceptable salts, esters and prodrugs thereof; wherein A, U, V, W, X and Z are substituents. The present invention also relates to methods for using such compounds.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. patent applicationSer. No. 10/821,243, filed Apr. 7, 2004, which claims the benefit ofU.S. provisional application 60/461,271, filed Apr. 7, 2003; U.S.provisional application 60/463,171, filed Apr. 15, 2003; U.S.provisional application 60/519,535, filed Nov. 12, 2003; and U.S.provisional application 60/532,727, filed Dec. 23, 2003. The contents ofthese documents are incorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to substituted quinobenzoxazines analogs, andmethods of using such compounds.

BACKGROUND

Quadruplexes can form in certain purine-rich strands of nucleic acids.In duplex nucleic acids, certain purine rich strands are capable ofengaging in a slow equilibrium between a typical duplex helix structureand in unwound and non-B-form regions. These unwound and non-B forms canbe referred to as “paranemic structures.” Some forms are associated withsensitivity to S1nuclease digestion, which can be referred to as“nuclease hypersensitivity elements” or “NHEs.” A quadruplex is one typeof paranemic structure and certain NHEs can adopt a quadruplexstructure. Considerable circumstantial evidence suggests that quadruplexstructures can exist in vivo in specific regions of the genome,including the telomeric ends of chromosomes and oncogene regulatoryregions. (Han, et al., Trends Pharm. Sci. (2000) 21:136-142). Thus,quadruplex forming regions of DNA may be used as molecular targets foranticancer agents.

SUMMARY OF THE INVENTION

Compounds described herein interact with regions of DNA that can formquadruplexes and act as tumor suppression agents with reduced sideeffects. Such compounds reduce expression of highly proliferate genesand are utilized for treating cancers. Furthermore, the compounds mayalso exhibit antibacterial or antiviral activity, and may be used fortreating bacterial and viral infections.

Various embodiments of the present invention are described below. Thepresent invention encompasses other compounds having formula 1, withsubstituents independently selected from compounds in Tables 1-3. Thus,the present invention is not limited to the specific combination ofsubstituents described in various embodiments below.

The compounds have the general formula:

and pharmaceutically acceptable salts, esters and prodrugs thereof;

-   -   wherein V is H, halo, or NR¹R²;    -   A is H, fluoro, or NR¹ ₂;    -   Z is O, S, NR¹ or CH₂;    -   U is OR² or NR¹R²;    -   X is OR², NR¹R², halo, azido, or SR²;    -   n is 1-3;    -   wherein in NR¹R², R¹ and R² may form a double bound or a ring,        each of which is optionally substituted;    -   R¹ is H or a C₁₋₆ alkyl;    -   R² is H or a C₁₋₁₀ alkyl or C₂₋₁₀ alkenyl optionally containing        one or more non-adjacent heteroatoms selected from N, O, and S,        and optionally substituted with a carbocyclic or heterocyclic        ring; or R² is an optionally substituted heterocyclic ring, aryl        or heteroaryl;    -   W is selected from the group consisting of        wherein Q, Q¹, Q², and Q³ are independently CH or N;    -   Y is independently O, CH, ═O or NR¹;    -   and R⁵ is a substituent at any position on the fused ring; and        is H, OR², Cl₁₋₆ alkyl, C₂₋₆ alkenyl, each optionally        substituted by halo, ═O or one or more heteroatoms; or R⁵ is an        inorganic substituent; or two adjacent R⁵ is linked to obtain a        5-6 membered substituted or unsubstituted carbocyclic or        heterocyclic ring, optionally fused to an additional substituted        or unsubstituted carbocyclic or heterocyclic ring;    -   provided that U is not OR¹ when X is pyrrolidinyl; A is F; Z is        O; and W is naphthalenyl or phenylene;    -   U is not morpholinyl or 2,4-difluoroaniline when X is F or        pyrrolidinyl; A is F; Z is O; and W is phenylene; and    -   further provided that if U is OH, then W represents multiple        fused aromatic rings and X is not halo; and X is NH₂, or a        moiety that does not contain N, or contains more than 6 carbons.

In the above formula 1, A and X may independently be halo. In oneexample, A and X may independently be fluoro.

In the above formula, V may be H. Alternatively, V may be NH₂ or acompound having the formula NR¹(CR¹ ₂)_(n)−NR³R⁴;

-   -   wherein R¹ and R³ are independently H or C₁₋₆ alkyl;    -   n is 1-6; and    -   R⁴ is H, C₁₋₆ alkyl optionally substituted with a carbocyclic or        heterocyclic ring, or aryl; and    -   wherein in NR³R⁴, R³ and R⁴ may form an optionally substituted        ring.

In the above formula 1, U and X may independently be NR¹R². In oneexample, R¹ is H and R² is a C₁₋₁₀ alkyl optionally containing one ormore heteroatoms, and optionally substituted with a C₃₋₆ cycloalkyl,aryl or a 5-14 membered heterocyclic ring containing one or more N, O orS. In another example, R¹ is H and R² is an aryl or a 5-14 memberedheterocyclic ring containing one or more N, O or S, each optionallysubstituted with an amino or another heterocyclic ring. In yet anotherexample, R¹ and R² in NR¹R² form an optionally substituted 5-14 memberedring containing one or more N, O or S. In particular examples, NR¹R² ismorpholine, thiomorpholine, piperazine, piperidine or diazepine.

In the above formula 1, U and X may independently have the formulaNR¹—(CR¹ ₂)_(n)-NR³R⁴   (2)wherein R¹ and R³ are independently H or C₁₋₆ alkyl;

-   -   n is 1-6; and    -   R⁴ is H or a C₁₋₁₀ alkyl or C₂₋₁₀ alkenyl optionally containing        one or more non-adjacent heteroatoms selected from N, O and S,        and optionally substituted with a carbocyclic or heterocyclic        ring; and    -   wherein in NR³R⁴, R³ and R⁴ may form an optionally substituted        ring.

In the above formula 2, n may be 2-3. In one example, NR³R⁴ is anacyclic amine, or guanidinyl or a tautomer thereof; or R³ and R⁴optionally form a substituted ring containing one or more N, O or S. Inparticular examples, NR³R⁴ is morpholine, thiomorpholine, imidazole,pyrrolidine, piperazine, pyridine or piperidine.

In the above formula 1, X may be NR¹R²; and U has the formulaNR¹—(CR¹ ₂)_(n)—NR³R⁴   (2)

-   -   wherein R¹ and R² are as defined in claim 1;    -   R³ is H or C₁₋₆ alkyl;    -   n is 1-6; and

R⁴ is H or a C₁₋₁₀ alkyl or C₂₋₁₀ alkenyl optionally containing one ormore non-adjacent heteroatoms selected from N, O and S, and optionallysubstituted with a carbocyclic or heterocyclic ring; and

-   -   wherein in NR¹R² and NR³R⁴, R¹ and R², and R³ and R⁴ each        independently may form a substituted ring.

In the above formula, where X is NR¹R² and U has the formula NR¹—(CR¹₂)_(n)—NR³R⁴ (2), R¹ and R² in NR¹R², and R³ and R⁴ in NR³R⁴ each mayindependently form ring containing one or more N, O or S. For example, Xis optionally substituted with amino, carbamate, a C₁₋₁₀ alkylcontaining one or more non-adjacent N, O or S, and optionallysubstituted with a heterocyclic ring; aryl or a saturated or unsaturatedheterocyclic ring, each of which is optionally substituted. In oneexample, X and NR³R⁴ are independently morpholine, thiomorpholine,imidazole, pyrrolidine, piperazine, pyridine or piperidine. In oneexample, X and NR³R⁴ are independently pyrrolidine. In another example,X is pyrrolidine substituted with pyrazine. In this example, V is H; Ais fluoro; and W is naphthalenyl.

Examples of 5-6 membered heterocyclic rings include but are not limitedto tetrahydrofuran, 1,3-dioxolane, 2,3-dihydrofuran, tetrahydropyran,benzofuran, isobenzofuran, 1,3-dihydro-isobenzofuran, isoxazole,4,5-dihydroisoxazole, piperidine, pyrrolidine, pyrrolidin-2-one,pyrrole, pyridine, pyrimidine, octahydro-pyrrolo[3,4-b]pyridine,piperazine, pyrazine, morpholine, thiomorpholine, imidazole,imidazolidine-2,4-dione, benzimidazole, 1,3-dihydrobenzimidazol-2-one,indole, thiazole, benzothiazole, thiadiazole, thiophene,tetrahydro-thiophene 1,1-dioxide, diazepine, triazole, guanidine,diazabicyclo[2.2.1]heptane, 2,5-diazabicyclo[2.2.1]heptane, and2,3,4,4a,9,9a-hexahydro-1H-β-carboline.

In the above formula 1, W may be benzene, pyridine, biphenyl,naphthalene, phenanthrene, quinoline, isoquinoline, quinazoline,cinnoline, phthalazine, quinoxaline, indole, benzimidazole, benzoxazole,benzthiazole, benzofuran, anthrone, xanthone, acridone, fluorenone,carbazolyl, pyrimido[4,3-b]furan, pyrido[4,3-b]indole,pyrido[2,3-b]indole, dibenzofuran, acridine or acridizine.

In the above formula 1, U may be OR² and R² is a C₁₋₆ alkyl optionallysubstituted with a carbocyclic or heterocyclic ring.

In the above formula 1, each optionally substituted moiety issubstituted with one or more halo, OR², NR¹R², carbamate, C₁₋₁₀ alkyl,C₂₋₁₀ alkenyl, each optionally substituted by halo, ═O, aryl or one ormore heteroatoms; inorganic substituents, aryl, carbocyclic or aheterocyclic ring.

The compounds of the present invention may be chiral. As used herein, achiral compound is a compound that is different from its mirror image,and has an enantiomer. Methods of synthesizing chiral compounds andresolving a racemic mixture of enantiomers are well known to thoseskilled in the art. See, e.g., March, “Advanced Organic Chemistry,” JohnWiley and Sons, Inc., New York, (1985), which is incorporated herein byreference.

The present invention also provides pharmaceutical compositionscomprising compounds having formula 1, and a pharmaceutically acceptableexcipient. In one example, the pharmaceutical composition comprisesabout 2% w/w of a compound having formula (1), about 4% mannitol, andabout 0.5% sucrose.

The present invention also provides a compound having formula (1A),

and pharmaceutically acceptable salts, esters and prodrugs thereof. Thecompound may be chiral. The present invention also provides apharmaceutical composition comprising a compound having formula 1A and apharmaceutically acceptable excipient.

The compounds and pharmaceutical compositions of the present inventionmay be formulated for any suitable route of administration. For example,the compounds and pharmaceutical compositions may be formulated forinjection. Other suitable routes of administration include but are notlimited to oral, parenteral, intravenous, intramuscular, topical,subcutaneous routes, and other routes known to the skilled person.

In one example, the pharmaceutical composition comprises about 2% w/w ofthe compound having formula 1A. The pharmaceutical composition mayfurther comprise about 4% w/w mannitol, 0.5% sucrose, or both. In otherexamples, the pharmaceutical composition has a pH of about 3.5.

In one particular embodiment, the pharmaceutical composition comprisesabout 2% w/w of a compound having formula 1A, 4% w/w mannitol, and 0.5%sucrose. In one example, the pharmaceutical formulation may be aqueous,injectable or both. For injectable formulations, water may be added tothe final weight.

The compounds and pharmaceutical compositions of the present inventionmay be administered with another therapeutic agent. For example, thecompounds and pharmaceutical compositions of the present invention maybe administered with a chemotherapeutic agent. Examples ofchemotherapeutic agents include but are not limited to anti-infectiveagents, antihelmintics, antiprotozoal agents, antimalarial agents,antiamebic agents, sulfonamides, antimycobacterial drugs, or antiviralchemotherapeutics. Chemotherapeutic agents may also be antineoplasticagents or cytotoxic drugs, such as alkylating agents, plant alkaloids,antimetabolites, antibiotics, and other anticellular proliferativeagents. In other examples, the compounds and pharmaceutical formulationsof the present invention may be administered with hepatic enzymeinducers or inhibitors.

Furthermore, the present invention also provides methods forameliorating a cell proliferative disorder, comprising administering toa subject in need thereof an effective amount of a compound havingformula 1 or 1A, or pharmaceutical compositions thereof, therebyameliorating said cell-proliferative disorder. In one example, the cellproliferative disorder is cancer. In another example, cell proliferationis reduced, or cell death is induced. The subject may be human oranimal.

The present invention also provides methods for reducing cellproliferation or inducing cell death, comprising contacting a systemwith an effective amount of a compound having formula 1 or 1A, orpharmaceutical compositions thereof, thereby reducing cell proliferationor inducing cell death in said system. The system may be a cell ortissue.

The present invention further provides methods for reducing microbialtiters, comprising contacting a system with an effective amount of acompound having formula 1 or 1A, or pharmaceutical compositions thereof,thereby reducing microbial titers. The system may be a cell or tissue.In one example, the microbial titers are viral, bacterial or fungaltiters.

Further, the present invention provides methods for ameliorating amicrobial infection, comprising administering to a subject in needthereof an effective amount of a compound formula 1 or 1A, orpharmaceutical compositions thereof, thereby ameliorating said microbialinfection. The subject may be human or animal. In one example, themicrobial infection is viral, bacterial or fungal.

DESCRIPTION OF THE FIGURES

FIG. 1 shows antitumor activity of a compound having formula 1 tested ina Ramos xenograft model.

FIG. 2 shows antitumor activity of a compound having formula 1 tested inHCT 116.

FIG. 3 shows a stop assay as a first pass screen for oncogeneinhibitors.

FIG. 4 shows the electrophoretogram for CX-3543, mAMSA, distamycins,mithramycin, actinomycin D, and ciprofloxacin.

FIG. 5 shows the quadruplex/binding selectivity of CX-3543.

FIG. 6 shows the Quadrome footprint for CX-3543.

FIG. 7 shows the topoisomerase II assay for CX-3543.

FIG. 8 shows the pharmacokinetic profile of CX-3543.

FIG. 9 shows the antitumor activity of CX-3543 in HCT-116 colorectalcancer xenograft model.

FIG. 10 shows the antitumor activity of CX-3543 in PC3 prostate cancerxenograft model.

DESCRIPTION OF THE INVENTION

The present invention relates to quinoline derivatives having formula 1,and pharmaceutically acceptable salts, esters, and prodrugs thereof. Inparticular embodiments, the compounds interact with regions of DNA thatcan form quadruplexes. The present invention also relates to methods fortreating cancer, bacterial and viral infections using such compounds.

Because regions of DNA that can form quadruplexes are regulators ofbiological processes such as oncogene transcription, modulators ofquadruplex biological activity can be utilized as cancer therapeutics.Molecules that interact with regions of DNA that can form quadruplexescan exert a therapeutic effect on certain cell proliferative disordersand related conditions. Particularly, abnormally increased oncogeneexpression can cause cell proliferative disorders and quadruplexstructures typically down-regulate oncogene expression. Examples ofoncogenes include but are not limited to MYC, HIF, VEGF, ABL, TGF,PDGFA, MYB, SPARC, HUMTEL, HER, VAV, RET, H-RAS, EGF, SRC, BCL1, BCL2,and other oncogenes known to one of skill in the art.

Molecules that bind to regions of DNA that can form quadruplexes canexert a biological effect according to different mechanisms, whichinclude for example, stabilizing a native quadruplex structure,inhibiting conversion of a native quadruplex to duplex DNA by blockingstrand cleavage, and stabilizing a native quadruplex structure having aquadruplex-destabilizing nucleotide substitution and other sequencespecific interactions. Thus, compounds that bind to regions of DNA thatcan form quadruplexes described herein may be administered to cells,tissues, or organisms for the purpose of down-regulating oncogenetranscription and thereby treating cell proliferative disorders. Theterms “treatment” and “therapeutic effect” as used herein refer toreducing or stopping a cell proliferation rate (e.g., slowing or haltingtumor growth) or reducing the number of proliferating cancer cells(e.g., removing part or all of a tumor).

Determining whether the biological activity of native DNA that can formquadruplexes is modulated in a cell, tissue, or organism can beaccomplished by monitoring quadruplex biological activity. Quadruplexforming regions of DNA biological activity may be monitored in cells,tissues, or organisms, for example, by detecting a decrease or increaseof gene transcription in response to contacting the quadruplex formingDNA with a molecule. Transcription can be detected by directly observingRNA transcripts or observing polypeptides translated by transcripts,which are methods well known in the art.

Molecules that interact with quadruplex forming DNA and quadruplexforming nucleic acids can be utilized to treat many cell proliferativedisorders. Cell proliferative disorders include, for example, colorectalcancers and hematopoietic neoplastic disorders (i.e., diseases involvinghyperplastic/neoplastic cells of hematopoietic origin such as thosearising from myeloid, lymphoid or erythroid lineages, or precursor cellsthereof). The diseases can arise from poorly differentiated acuteleukemias, e.g., erythroblastic leukemia and acute megakaryoblasticleukemia. Additional myeloid disorders include, but are not limited to,acute promyeloid leukemia (APML), acute myelogenous leukemia (AML) andchronic myelogenous leukemia (CML) (Vaickus, Crit. Rev. inOncol/Hemotol. 11:267-297 (1991)). Lymphoid malignancies include, butare not limited to acute lymphoblastic leukemia (ALL), which includesB-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL),prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) andWaldenstrom's macroglobulinemia (WM). Additional forms of malignantlymphomas include, but are not limited to non-Hodgkin lymphoma andvariants thereof, peripheral T cell lymphomas, adult T cellleukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), largegranular lymphocytic leukemia (LGF), Hodgkin's disease andReed-Sternberg disease. Cell proliferative disorders also includecancers of the colorectum, breast, lung, liver, pancreas, lymph node,colon, prostate, brain, head and neck, skin, liver, kidney, and heart.Compounds that interact with regions of DNA that can form quadruplexesalso can be utilized to target cancer related processes and conditions,such as increased angiogenesis, by inhibiting angiogenesis in a subject.

The present invention provides a method for reducing cell proliferationor for treating or alleviating cell proliferative disorders, comprisingcontacting a system having a DNA capable of forming a quadruplex with acompound having formula 1. The system may be a group of cells or one ormore tissues. In one embodiment, the system is a subject in need of atreatment of a cell proliferative disorder (e.g., a mammal such as amouse, rat, monkey, or human). The present invention also provides amethod for treating colorectal cancer by administering a compound thatinteracts with a c-MYC quadruplex forming region to a subject in needthereof, thereby reducing the colorectal cancer cell proliferation.Furthermore, the present invention provides a method for inhibitingangiogenesis and optionally treating a cancer associated withangiogenesis, comprising administering a compound that interacts with avascular endothelial growth factor (VEGF) quadruplex forming region to asubject in need thereof, thereby reducing angiogenesis and optionallytreating a cancer associated with angiogenesis.

As used herein, the term “alkyl” refers to a carbon-containing compound,and encompasses compounds containing one or more heteroatoms. The term“alkyl” also encompasses compounds substituted with one or morenon-interfering substituents. Examples of non-interfering substituentsinclude but are not limited to OR¹, amino, amido, halo, ═O, aryl,heterocyclic groups, or inorganic substituents, and other substituentsthat do not interfere with the activity of the compound.

As used herein, the term “carbocycle” refers to a cyclic compoundcontaining only carbon atoms in the ring.

As used herein, the term “heterocycle” refers to a cyclic compoundcomprising a heteroatom, including monocyclic or bicyclic heterocycles.As used herein, the term “heteroatom” refers to any atom that is notcarbon or hydrogen, such as nitrogen, oxygen or sulfur. Examples ofheterocycles include but are not limited to oxirane, oxetane, pyran,tetrahydropyran, dioxane, lactones, aziridine, azetidine, pyrrolidine,piperidine, morpholine, lactams, and tetrahydrofuran.

As used herein, the term “bicyclic compound” refers to a compound havingtwo rings which share a pair of bridgehead carbon atoms. Examples ofbicyclic compounds include but are not limited to decalin, norbornane,camphor, and diazabicyclo[2.2.1]heptane.

As used herein, the terms “heteroaryl” or “heteroaromatic” refer to anaromatic heterocycle. Examples of heteroaryls include but are notlimited to furan, pyrrole, pyridine, pyrimidine, imidazole, or triazole.

The terms “treat,” “treatment” and “therapeutic effect” as used hereinrefer to reducing or stopping a cell proliferation rate (e.g., slowingor halting tumor growth) or reducing the number of proliferating cancercells (e.g., removing part or all of a tumor). These terms also areapplicable to reducing a titre of a microorganism in a system (i.e.,cell, tissue, or subject) infected with a microorganism, reducing therate of microbial propagation, reducing the number of symptoms or aneffect of a symptom associated with the microbial infection, and/orremoving detectable amounts of the microbe from the system. Examples ofmicroorganism include but are not limited to virus, bacterium andfungus.

Compounds that interact with quadruplex forming regions of DNA can alsobe used to reduce a microbial infection, such as a viral infection.Retroviruses offer a wealth of potential targets for G-quadruplextargeted therapeutics. G-quadruplex structures have been implicated asfunctional elements in at least two secondary structures formed byeither viral RNA or DNA in HIV, the dimer linker structure (DLS) and thecentral DNA flap (CDF). Additionally, DNA aptamers which are able toadopt either inter- or intramolecular quadruplex structures are able toinhibit viral replication. In one example, DNA aptamers are able toinhibit viral replication by targeting the envelope glycoprotein(putatively). In another example, DNA aptamers inhibit viral replicationby targeting the HIV-integrase respectively, suggesting the involvementof native quadruplex structures in interaction with the integraseenzyme.

Dimer linker structures, which are common to all retroviruses, serve tobind two copies of the viral genome together by a non-covalentinteraction between the two 5′ ends of the two viral RNA sequences. Thegenomic dimer is stably associated with the gag protein in the maturevirus particle. In the case of HIV, the origin of this non-covalentbinding may be traced to a 98 base-pair sequence containing several runsof at least two consecutive guanines (e.g., the 3′ for the formation ofRNA dimers in vitro). An observed cation (potassium) dependence for theformation and stability of the dimer in vitro, in addition to thefailure of an antisense sequence to effectively dimerize, has revealedthe most likely binding structure to be an intermolecular G-quadruplex.

Prior to integration into the host genome, reverse transcribed viral DNAforms a pre-integration complex (PIC) with at least two major viralproteins, integrase and reverse transcriptase, which is subsequentlytransported into the nucleus. The Central DNA Flap (CDF) refers to99-base length single-stranded tail of the +strand, occurring near thecenter of the viral duplex DNA, which is known to a play a role in thenuclear import of the PIC. Oligonucleotide mimics of the CDF have beenshown to form intermolecular G-quadruplex structures in cell-freesystems.

Thus, compounds that recognize quadruplex forming regions can be used tostabilize the dimer linker structure and thus prevent de-coupling of thetwo RNA strands. Also, by binding to the quadruplex structure formed bythe CDF, protein recognition and/or binding events for nuclear transportof the PIC may be disrupted. In either case, a substantial advantage canexist over other anti-viral therapeutics. Current Highly ActiveAnti-Retroviral Therapeutic (HAART) regimes rely on the use ofcombinations of drugs targeted towards the HIV protease and HIVintegrase. The requirement for multi-drug regimes is to minimize theemergence of resistance, which will usually develop rapidly when agentsare used in isolation. The source of such rapid resistance is theinfidelity of the reverse transcriptase enzyme which makes a mutationapproximately once in every 10,000 base pairs. An advantage of targetingviral quadruplex structures over protein targets, is that thedevelopment of resistance is slow or is impossible. A point mutation ofthe target quadruplex can compromise the integrity of the quadruplexstructure and lead to a non-functional copy of the virus. A singletherapeutic agent based on this concept may replace the multiple drugregimes currently employed, with the concomitant benefits of reducedcosts and the elimination of harmful drug/drug interactions.

The present invention provides a method for reducing a microbial titerin a system, comprising contacting a system having a native DNAquadruplex forming region with a compound having formula 1. The systemmay be one or more cells or tissues. Examples of microbial titersinclude but are not limited to viral, bacterial or fungal titers. In aparticular embodiment, the system is a subject in need of a treatmentfor a viral infection (e.g., a mammal such as a mouse, rat, monkey, orhuman). Examples of viral infections include infections by a hepatitisvirus (e.g., hepatitis B or C), human immunodeficiency virus (HIV),rhinovirus, herpes-zoster virus (VZV), herpes simplex virus (e.g., HSV-1or HSV-2), cytomegalovirus (CMV), vaccinia virus, influenza virus,encephalitis virus, hantavirus, arbovirus, West Nile virus, humanpapilloma virus (HPV), Epstein-Barr virus, and respiratory syncytialvirus. The present invention also provides a method for treating HIVinfection by administering a compound having formula 1 to a subject inneed thereof, thereby reducing the HIV infection.

Identifying Compounds That Can Bind to Quadruplex Forming Regions of DNA

Compounds described herein are identified as compounds that can bind toquadruplex forming regions of DNA where a biological activity of thisregion, often expressed as a “signal,” produced in a system containingthe compound is different than the signal produced in a system notcontaining the compound. While background signals may be assessed eachtime a new molecule is probed by the assay, detecting the backgroundsignal is not required each time a new molecule is assayed.

Examples of quadruplex forming nucleic acid sequences are set forth inthe following Table A: TABLE A SEQ SEQUENCE ID NO ORIGINTG₄AG₃TG₄AG₃TG₄AAGG 1 CMYC GGGGGGGGGGGGGCGGGGGCGGGGGCGGGGGAGGGGC 2 PDGFAG₈ACGCG₃AGCTG₅AG₃CTTG₄CCAG₃CG₄CGCTTAG₅ 3 PDGFB/c -sisAGGAAGGGGAGGGCCGGGGGGAGGTGGC 4 CABL AGGGGCGGGGCGGGGCGGGGGC 5 RETGGGAGGAAGGGGGCGGGAGCGGGGC 6 BCL-2 GGGGGGCGGGGGCGGGCGCAGGGGGAGGGGGC 7Cyclin D1/BCL-1 CGGGGCGGGGCGGGGGCGGGGGC 8 H-RASAGAGGAGGAGGAGGTCACGGAGGAGGAGGAGAAGGAGGAGGAGGAA 9 CMYB (GGA)₄ 10 VAVAGAGAAGAGGGGAGGAGGAGGAGGAGAGGAGGAGGCGC 11 HMGA2 GGAGGGGGAGGGG 12 CPIMAGGAGAAGGAGGAGGTGGAGGAGGAGG 13 HER2/neuAGGAGGAGGAGAATGCGAGGAGGAGGGAGGAGA 14 EGFRGGGGCGGGCCGGGGGCGGGGTCCCGGCGGGGCGGAG 15 VEGF CGGGAGGAGGAGGAAGGAGGAAGCGCG16 CSRC

In addition to determining whether a test molecule or test nucleic acidgives rise to a different signal, the affinity of the interactionbetween the nucleic acid and the compound may be quantified. IC₅₀,K_(d), or K_(i) threshold values may be compared to the measured IC₅₀ orK_(d) values for each interaction, and thereby identify a test moleculeas a quadruplex interacting molecule or a test nucleic acid as aquadruplex forming nucleic acid. For example, IC₅₀ or K_(d) thresholdvalues of 10 μM or less, 1 μM or less, and 100 nM or less are oftenutilized. In another example, threshold values of 10 nM or less, 1 nM orless, 100 pM or less, and 10 pM or less may be utilized to identifyquadruplex interacting molecules and quadruplex forming nucleic acids.

Many assays are available for identifying compounds that have affinityfor quadruplex forming regions of DNA. In some of these assays, thebiological activity is the quadruplex nucleic acid binding to a compoundand binding is measured as a signal. In other assays, the biologicalactivity, is a polymerase arresting function of a quadruplex and thedegree of arrest is measured as a decrease in a signal. In certainassays, the biological activity is transcription and transcriptionlevels can be quantified as a signal. In another assay, the biologicalactivity is cell death and the number of cells undergoing cell death isquantified. Another assay monitors proliferation rates of cancer cells.Examples of assays are fluorescence binding assays, gel mobility shiftassays (see, e.g., Jin & Pike, Mol. Endocrinol. (1996)10:196-205),polymerase arrest assays, transcription reporter assays, cancer cellproliferation assays, and apoptosis assays (see, e.g., AmershamBiosciences (Piscataway, N.J.)), and embodiments of such assays aredescribed hereafter. Also, topoisomerase assays can be utilized todetermine whether the quadruplex interacting molecules have atopoisomerase pathway activity (see, e.g. TopoGEN, Inc. (Columbus,Ohio)).

Gel Electrophoretic Mobility Shift Assay (EMSA)

An EMSA is useful for determining whether a nucleic acid forms aquadruplex and whether a nucleotide sequence isquadruplex-destabilizing. EMSA is conducted as described previously (Jin& Pike, Mol. Endocrinol. 10: 196-205 (1996)) with minor modifications.Generally, synthetic single-stranded oligonucleotides are labeled in the5′-terminus with T4-kinase in the presence of [γ³²P] ATP (1,000mCi/mmol, Amersham Life Science) and purified through a sephadex column.³²P-labeled oligonucleotides (˜30,000 cpm) are then incubated with orwithout various concentrations of a testing compound in 20 μl of abuffer containing 10 mM Tris pH 7.5, 100 mM KCl, 5 mM dithiothreitol,0.1 mM EDTA, 5 mM MgCl₂, 10% glycerol, 0.05% Nonedit P-40, and 0.1 mg/mlof poly(dI-dC) (Pharmacia). After incubation for 20 minutes at roomtemperature, binding reactions are loaded on a 5% polyacrylamide gel in0.25× Tris borate-EDTA buffer (0.25×TBE, 1×TBE is 89 mM Tris-borate, pH8.0, 1 mM EDTA). The gel is dried and each band is quantified using aphosphoimager.

DMS Methylation Protection Assay

Chemical footprinting assays are useful for assessing quadruplexstructure. Quadruplex structure is assessed by determining whichnucleotides in a nucleic acid are protected or unprotected from chemicalmodification as a result of being inaccessible or accessible,respectively, to the modifying reagent. A DMS methylation assay is anexample of a chemical footprinting assay. In such an assay, bands fromEMSA are isolated and subjected to DMS-induced strand cleavage. Eachband of interest is excised from an electrophoretic mobility shift geland soaked in 100 mM KCl solution (300 μl) for 6 hours at 4° C. Thesolutions are filtered (microcentrifuge) and 30,000 cpm (per reaction)of DNA solution is diluted further with 100 mM KCl in 0.1×TE to a totalvolume of 70 μl (per reaction). Following the addition of 1 μl salmonsperm DNA (0.1 μg/μl), the reaction mixture is incubated with 1 μl DMSsolution (DMS:ethanol; 4:1; v:v) for a period of time. Each reaction isquenched with 18 μl of stop buffer (b-mercaptoethanol:water:NaOAc (3 M);1:6:7; v:v:v). Following ethanol precipitation (twice)-and piperidinecleavage, the reactions are separated on a preparative gel (16%) andvisualized on a phosphoimager.

Polymerase Arrest Assay

An arrest assay includes a template nucleic acid, which may comprise aquadruplex forming sequence, and a primer nucleic acid which hybridizesto the template nucleic acid 5′ of the quadruplex-forming sequence. Theprimer is extended by a polymerase (e.g., Taq polymerase), whichadvances from the primer along the template nucleic acid. In this assay,a quadruplex structure can block or arrest the advance of the enzyme,leading to shorter transcription fragments. Also, the arrest assay maybe conducted at a variety of temperatures, including 45° C. and 60° C.,and at a variety of ion concentrations.

An example of the Taq polymerase stop assay is described in Han, et al.,Nucl. Acids Res. (1999) 27:537-542, which is a modification of that usedby Weitzmann, et al., J. Biol. Chem. (1996) 271 :20958-20964. Briefly, areaction mixture of template DNA (50 nM), Tris-HCl (50 mM), MgCl₂ (10mM), DTT (0.5 mM), EDTA (0.1 mM), BSA (60 ng), and 5′-end-labeledquadruplex nucleic acid (˜18 nM) is heated to 90° C. for 5 minutes andallowed to cool to ambient temperature over 30 minutes. Taq Polymerase(1 μl) is added to the reaction mixture, and the reaction is maintainedat a constant temperature for 30 minutes. Following the addition of 10μl stop buffer (formamide (20 ml), 1 M NaOH (200 μl), 0.5 M EDTA (400μl), and 10 mg bromophenol blue), the reactions are separated on apreparative gel (12%) and visualized on a phosphoimager. Adeninesequencing (indicated by “A” at the top of the gel) is performed usingdouble-stranded DNA Cycle Sequencing System from Life Technologies. Thegeneral sequence for the template strands isTCCAACTATGTATAC-INSERT-TTAGCGACACGCAATTGCTATAGTGAGTCGTATTA, where“INSERT” refers to a nucleic acid sequence comprising a quadruplexforming sequence (See e.g., Table A). Bands on the gel that exhibitslower mobility are indicative of quadruplex formation.

High Throughput Polymerase Arrest Assay

A high throughput polymerase arrest assay has been developed. The assaycomprises contacting a template nucleic acid, often DNA, with a primer,which also is often DNA; contacting the primer/template complex with acompound described herein (also referred to as a “test compound”);contacting the primer/template complex with a polymerase; and separatingreaction products. The assay often includes the step of denaturing theprimer/template complex mixture and then renaturing the complex, whichoften is carried out before a test molecule is added to the system.Multiple assays often are carried out using varying concentrations of atest compound, such that an IC₅₀ value can be obtained, for example. Thereaction products often include extended primers of different lengths.Where a test compound does not significantly interact with a quadruplexstructure in the template, the primer often is extended to the end ofthe template.

Where a test compound significantly interacts with a quadruplexstructure in the template, the primer often is extended only to thequadruplex structure in the template and no further. Thus, the reactionmixture often includes at least two reaction products when a testcompound interacts with a quadruplex structure in the template, onehaving a completely extended primer and one having an incompletelyextended primer, and these two reaction products are separated. Theproducts may be separated using any convenient separation method, suchas mass spectrometry and in one embodiment, capillary electrophoresis.

The reaction products often are identified by detecting a detectablelabel linked to the primer. The detectable label may be non-covalentlylinked to the 5′ end of the primer (e.g., a biotin molecule covalentlylinked to the 5′ end of the primer which is non-covalently linked to anavidin molecule joined to a detectable label). The detectable label maybe joined to the primer at any stage of the assay, sometimes before theprimer is added to the system, after the primer is extended, or afterthe products are separated. The detectable label often is covalentlylinked to the primer using a procedure selected based upon the nature ofthe chemical groups in the detectable label.

Many methods for covalently linking detectable labels to nucleic acidsare available, such as chemically coupling an allylamine-derivatizednucleotide to a succinimidyl-ester derivative of a detectable label, andthen generating a primer using the labeled nucleotide. (See, e.g.,Nature Biotech (2000) 18:345-348 and http addressinfo.med.yale.edu/genetics/ward/tavi/n_coupling.html). A spacer (oftenbetween 5-16 carbon atoms long) sometimes is incorporated between thedetectable label and the nucleotide. Any convenient detectable label maybe utilized, including but not limited to a radioactive isotope (e.g.,¹²⁵I, ¹³¹I, ³⁵S, ³²p, ¹⁴C or ³H); a light scattering label (e.g., aspherical gold or silver label; Genicon Sciences Corporation, San Diego,Calif. and U.S. Pat. No. 6,214,560); an enzymic or protein label (e.g.,GFP or peroxidase); or another chromogenic label or dye sometimes isutilized. Often, a fluorescent label is utilized (e.g., amino-methylcoumarin (AMCA); diethyl aminomethyl coumarin (DEAC); cascade blue (CB);fluorescein isothiocyanate (FITC); Oregon green (OG); Alexa 488 (A488);rhodamine green (RGr); lanthanide chelate (e.g., europium),carboxy-rhodamine 6G (R6G); tetramethyl rhodamine (TAMRA); Texas Red(TxR); Cy3; Cy3.5; Cy5, Cy5.5 and carboxynaphtofluorescein (CNF),digoxigenin (DIG); and 2,4-dinitrophenyl (DNP)). Other fluorophores andattendant excitation and emission wavelengths are described in Anantha,et al., Biochemistry (1998) 37:2709-2714 and Qu & Chaires, MethodsEnzymol (2000) 321:353-369).

In an embodiment, a primer oligonucleotide covalently linked to afluorescent label is contacted with template DNA. The resulting complexis contacted with a test molecule and then contacted with a polymerasecapable of extending the primer. The reaction products then areseparated and detected by capillary electrophoresis. A longer primersequence was used for practicing this embodiment as compared toembodiments where the primer includes no covalently-linked fluorophoreor where capillary electrophoresis is not utilized for separation.Deoxynucleotides are added at any stage of the assay before theseparation, often when the primer is contacted with the template DNA.The template DNA/primer complex often is denatured (e.g., by increasingthe temperature of the system) and then renatured (e.g., by cooling thesystem) before a test compound is added).

The following is a specific example of the assay embodiment. A5′-fluorescent-labeled (FAM) primer (P45, 15 nM) was mixed with templateDNA (15 nM) in a Tris-HCL buffer (15 mM Tris, pH 7.5) containing 10 mMMgCl₂, 0.1 mM EDTA and 0.1 mM mixed deoxynucleotide triphosphates(dNTP's). The FAM-P45 primer (5′-6FAM-AGTCTGACTGACTGTACGTAGCTAATACGACTCACTATAGCAATT-3′) and the template DNA(5′-TCCAACTATGTATACTGGGGAGGGTGGGGAGGGTGGGGAAGGTTAGCGACACGCAATTGCTATAGTGAGTCGTATTAGCTACGTACAGTCAGTCAGACT-3′) were synthesized andHPLC purified by Applied Biosystems. The mixture was denatured at 95° C.for 5 minutes and, after cooling down to room temperature, was incubatedat 37° C. for 15 minutes.

After cooling down to room temperature, 1 mM KCl₂ and the test compound(various concentrations) were added and the mixture incubated for 15minutes at room temperature. The primer extension was performed byadding 10 mM KCl and Taq DNA Polymerase (2.5 U/reaction, Promega) andincubating at 70° C. for 30 minutes. The reaction was stopped by adding1 μl of the reaction mixture to 10 μl Hi-Di Formamide mixed and 0.25 μlLIZ 120 size standard. Hi-Di Formamide and LIZ120 size standard werepurchased from Applied Biosystems. The partially extended quadruplexarrest product was between 61 or 62 bases long and the full-lengthextended product was 99 bases long. The products were separated andanalyzed using capillary electrophoresis. Capillary electrophoresis wasperformed using an ABI PRISM 3100-Avant Genetic Analyzer. The assay wasperformed using compounds described above and results are shown inTable 1. μM concentrations reported in Table 1 are concentrations atwhich 50% of the DNA was arrested in the assay (i.e., the ratio ofshorter partially extended DNA (arrested DNA) to fill-length extendedDNA is 1:1).

Transcription Reporter Assay

In a transcription reporter assay, test quadruplex DNA is coupled to areporter system, such that a formation or stabilization of a quadruplexstructure can modulate a reporter signal. An example of such a system isa reporter expression system in which a polypeptide, such as luciferaseor green fluorescent protein (GFP), is expressed by a gene operablylinked to the potential quadruplex forming nucleic acid and expressionof the polypeptide can be detected. As used herein, the term “operablylinked” refers to a nucleotide sequence which is regulated by a sequencecomprising the potential quadruplex forming nucleic acid. A sequence maybe operably linked when it is on the same nucleic acid as the quadruplexDNA, or on a different nucleic acid. An exemplary luciferase reportersystem is described herein.

A luciferase promoter assay described in He, et al., Science (1998)281:1509-1512 often is utilized for the study of quadruplex formation.Specifically, a vector utilized for the assay is set forth in reference11 of the He, et al., document. In this assay, HeLa cells aretransfected using the lipofectamin 2000-based system (Invitrogen)according to the manufacturer's protocol, using 0.1 μg of pRL-TK(Renilla luciferase reporter plasmid) and 0.9 μg of thequadruplex-forming plasmid. Firefly and Renilla luciferase activitiesare assayed using the Dual Luciferase Reporter Assay System (Promega) ina 96-well plate format according to the manufacturer's protocol.

Circular Dichroism Assay

Circular dichroism (CD) is utilized to determine whether anothermolecule interacts with a quadruplex nucleic acid. CD is particularlyuseful for determining whether a PNA or PNA-peptide conjugate hybridizeswith a quadruplex nucleic acid in vitro. PNA probes are added toquadruplex DNA (5 μM each) in a buffer containing 10 mM potassiumphosphate (pH 7.2) and 10 or 250 mM KCl at 37° C. and then allowed tostand for 5 minutes at the same temperature before recording spectra. CDspectra are recorded on a Jasco J-715 spectropolarimeter equipped with athermoelectrically controlled single cell holder. CD intensity normallyis detected between 220 nm and 320 nm and comparative spectra forquadruplex DNA alone, PNA alone, and quadruplex DNA with PNA aregenerated to determine the presence or absence of an interaction (see,e.g., Datta, et al., JACS (2001) 123:9612-9619). Spectra are arranged torepresent the average of eight scans recorded at 100 nm/min.

Fluorescence Binding Assay

An example of a fluorescence binding assay is a system that includes aquadruplex nucleic acid, a signal molecule, and a test molecule. Thesignal molecule generates a fluorescent signal when bound to thequadruplex nucleic acid (e.g., N-methylmesoporphyrin IX (NMM)), and thesignal is altered when a test compound competes with the signal moleculefor binding to the quadruplex nucleic acid. An alteration in the signalwhen test molecule is present as compared to when test compound is notpresent identifies the test compound as a quadruplex interactingcompound.

50 μl of quadruplex nucleic acid or a nucleic acid not capable offorming a quadruplex is added in 96-well plate. A test compound also isadded in varying concentrations. A typical assay is carried out in 100μl of 20 mM HEPES buffer, pH 7.0, 140 mM NaCl, and 100 mM KCl. 50 μl ofthe signal molecule NMM then is added for a final concentration of 3 μM.NMM is obtained from Frontier Scientific Inc, Logan, Utah. Fluorescenceis measured at an excitation wavelength of 420 nm and an emissionwavelength of 660 nm using a FluroStar 2000 fluorometer (BMGLabtechnologies, Durham, N.C.). Fluorescence often is plotted as afunction of concentration of the test compound or quadruplex-targetednucleic acid and maximum fluorescent signals for NMM are assessed in theabsence of these molecules.

Cell Proliferation Assay

In a cancer cell proliferation assay, cell proliferation rates areassessed as a function of different concentrations of test compoundsadded to the cell culture medium. Any cancer cell type can be utilizedin the assay. In one embodiment, colon cancer cells are cultured invitro and test compounds are added to the culture medium at varyingconcentrations. A useful colon cancer cell line is colo320, which is acolon adenocarcinoma cell line deposited with the National Institutes ofHealth as accession number JCRB0225. Parameters for using such cells areavailable at the http addresscellbank.nihs.go.jp/cell/data/jcrb0225.htm.

Formulation of Compounds

As used herein, the term “pharmaceutically acceptable salts, esters andamides” includes but are not limited to carboxylate salts, amino acidaddition salts, esters and amides of the compounds, as well as thezwitterionic forms thereof, which are known to those skilled in the artas suitable for use with humans and animals. (See, e.g., Gerge, S. M.,et al., “Pharmaceutical Salts,” J. Pharm. Sci. (1977) 66:1-19, which isincorporated herein by reference.)

Any suitable formulation of the compounds described herein can beprepared. In cases where compounds are sufficiently basic or acidic toform stable nontoxic acid or base salts, administration of the compoundsas salts may be appropriate. Examples of pharmaceutically acceptablesalts are organic acid addition salts formed with acids that form aphysiological acceptable anion, for example, tosylate, methanesulfonate,acetate, citrate, malonate, tartarate, succinate, benzoate, ascorbate,a-ketoglutarate, and a-glycerophosphate. Suitable inorganic salts mayalso be formed, including hydrochloride, sulfate, nitrate, bicarbonate,and carbonate salts. Pharmaceutically acceptable salts are obtainedusing standard procedures well known in the art. For example,pharmaceutically acceptable salts may be obtained by reacting asufficiently basic compound such as an amine with a suitable acidaffording a physiologically acceptable anion. Alkali metal (e.g.,sodium, potassium or lithium) or alkaline earth metal (e.g., calcium)salts of carboxylic acids also are made.

A compound may be formulated as a pharmaceutical composition andadministered to a mammalian host in need of such treatment. In oneembodiment, the mammalian host is human. Any suitable route ofadministration may be used, including but not limited to oral,parenteral, intravenous, intramuscular, topical and subcutaneous routes.

In one embodiment, a compound is administered systemically (e.g.,orally) in combination with a pharmaceutically acceptable vehicle suchas an inert diluent or an assimilable edible carrier. They may beenclosed in hard or soft shell gelatin capsules, compressed intotablets, or incorporated directly with the food of the patient's diet.For oral therapeutic administration, the active compound may be combinedwith one or more excipients and used in the form of ingestible tablets,buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers,and the like. Such compositions and preparations should contain at least0.1% of active compound. The percentage of the compositions andpreparations may be varied and may conveniently be between about 2 toabout 60% of the weight of a given unit dosage form. The amount ofactive compound in such therapeutically useful compositions is such thatan effective dosage level will be obtained.

Tablets, troches, pills, capsules, and the like also may contain thefollowing: binders such as gum tragacanth, acacia, corn starch orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, fructose, lactose or aspartame or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring may be added. Whenthe unit dosage form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier, such as a vegetable oilor a polyethylene glycol. Various other materials may be present ascoatings or to otherwise modify the physical form of the solid unitdosage form. For instance, tablets, pills, or capsules may be coatedwith gelatin, wax, shellac or sugar and the like. A syrup or elixir maycontain the active compound, sucrose or fructose as a sweetening agent,methyl and propylparabens as preservatives, a dye and flavoring such ascherry or orange flavor. Any material used in preparing any unit dosageform is pharmaceutically acceptable and substantially non-toxic in theamounts employed. In addition, the active compound may be incorporatedinto sustained-release preparations and devices.

The active compound also may be administered intravenously orintraperitoneally by infusion or injection. Solutions of the activecompound or its salts may be prepared in a buffered solution, oftenphosphate buffered saline, optionally mixed with a nontoxic surfactant.Dispersions can also be prepared in glycerol, liquid polyethyleneglycols, triacetin, and mixtures thereof and in oils. Under ordinaryconditions of storage and use, these preparations contain a preservativeto prevent the growth of microorganisms. The compound is sometimesprepared as a polymatrix-containing formulation for such administration(e.g., a liposome or microsome). Liposomes are described for example inU.S. Pat. No. 5,703,055 (Felgner, et al.) and Gregoriadis, LiposomeTechnology vols. I to III (2nd ed. 1993).

The pharmaceutical dosage forms suitable for injection or infusion caninclude sterile aqueous solutions or dispersions or sterile powderscomprising the active ingredient that are adapted for the extemporaneouspreparation of sterile injectable or infusible solutions or dispersions,optionally encapsulated in liposomes. In all cases, the ultimate dosageform should be sterile, fluid and stable under the conditions ofmanufacture and storage. The liquid carrier or vehicle can be a solventor liquid dispersion medium comprising, for example, water, ethanol, apolyol (for example, glycerol, propylene glycol, liquid polyethyleneglycols, and the like), vegetable oils, nontoxic glyceryl esters, andsuitable mixtures thereof. The proper fluidity can be maintained, forexample, by the formation of liposomes, by the maintenance of theparticle size in the case of dispersions or by the use of surfactants.The prevention of the action of microorganisms can be brought about byvarious antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, buffers or sodium chloride. Prolonged absorption of theinjectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompound in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfilter sterilization. In the case of sterile powders for the preparationof sterile injectable solutions, the preferred methods of preparationare vacuum drying and the freeze drying techniques, which yield a powderof the active ingredient plus any additional desired ingredient presentin the previously sterile-filtered solutions.

For topical administration, the present compounds may be applied inliquid form. Compounds often are administered as compositions orformulations, in combination with a dermatologically acceptable carrier,which may be a solid or a liquid. Examples of useful dermatologicalcompositions used to deliver compounds to the skin are known (see, e.g.,Jacquet, et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No.4,992,478), Smith, et al. (U.S. Pat. No. 4,559,157) and Wortzman (U.S.Pat. No. 4,820,508).

Compounds may be formulated with a solid carrier, which include finelydivided solids such as talc, clay, microcrystalline cellulose, silica,alumina and the like. Useful liquid carriers include water, alcohols orglycols or water-alcohol/glycol blends, in which the present compoundscan be dissolved or dispersed at effective levels, optionally with theaid of non-toxic surfactants. Adjuvants such as fragrances andadditional antimicrobial agents can be added to optimize the propertiesfor a given use. The resultant liquid compositions can be applied fromabsorbent pads, used to impregnate bandages and other dressings, orsprayed onto the affected area using pump-type or aerosol sprayers.Thickeners such as synthetic polymers, fatty acids, fatty acid salts andesters, fatty alcohols, modified celluloses or modified mineralmaterials can also be employed with liquid carriers to form spreadablepastes, gels, ointments, soaps, and the like, for application directlyto the skin of the user.

Generally, the concentration of the compound in a liquid compositionoften is from about 0.1 wt % to about 25 wt %, sometimes from about 0.5wt % to about 10 wt %. The concentration in a semi-solid or solidcomposition such as a gel or a powder often is about 0.1 wt % to about 5wt %, sometimes about 0.5 wt % to about 2.5 wt %. A compound compositionmay be prepared as a unit dosage form, which is prepared according toconventional techniques known in the pharmaceutical industry. In generalterms, such techniques include bringing a compound into association withpharmaceutical carrier(s) and/or excipient(s) in liquid form or finelydivided solid form, or both, and then shaping the product if required.

In a particular example, the pharmaceutical composition comprises about2% w/w of a compound having formula (1), about 4% mannitol, and about0.5% sucrose. In particular examples, the formulation has a pH of about3.5. For injectable formulations, water may be added to the finalweight.

The compound composition may be formulated into any dosage form, such astablets, capsules, gel capsules, liquid syrups, soft gels,suppositories, and enemas. The compositions also may be formulated assuspensions in aqueous, non aqueous, or mixed media. Aqueous suspensionsmay further contain substances which increase viscosity, including forexample, sodium carboxymethylcellulose, sorbitol, and/or dextran. Thesuspension may also contain one or more stabilizers.

The amount of the compound, or an active salt or derivative thereof,required for use in treatment will vary not only with the particularsalt selected but also with the route of administration, the nature ofthe condition being treated and the age and condition of the patient andwill be ultimately at the discretion of the attendant physician orclinician.

A useful compound dosage often is determined by assessing its in vitroactivity in a cell or tissue system and/or in vivo activity in an animalsystem. For example, methods for extrapolating an effective dosage inmice and other animals to humans are known to the art (see, e.g., U.S.Pat. No. 4,938,949). Such systems can be used for determining the LD₅₀(the dose lethal to 50% of the population) and the ED₅₀ (the dosetherapeutically effective in 50% of the population) of a compound. Thedose ratio between a toxic and therapeutic effect is the therapeuticindex and it can be expressed as the ratio ED₅₀/LD₅₀. The compounddosage often lies within a range of circulating concentrations for whichthe ED₅₀ is associated with little or no toxicity. The dosage may varywithin this range depending upon the dosage form employed and the routeof administration utilized. For any compounds used in the methodsdescribed herein, the therapeutically effective dose can be estimatedinitially from cell culture assays. A dose sometimes is formulated toachieve a circulating plasma concentration range covering the IC₅₀(i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in in vitro assays,as such information often is used to more accurately determine usefuldoses in humans. Levels in plasma may be measured, for example, by highperformance liquid chromatography.

Another example of effective dose determination for a subject is theability to directly assay levels of “free” and “bound” compound in theserum of the test subject. Such assays may utilize antibody mimicsand/or “biosensors” generated by molecular imprinting techniques. Thecompound is used as a template, or “imprinting molecule”, to spatiallyorganize polymerizable monomers prior to their polymerization withcatalytic reagents. Subsequent removal of the imprinted molecule leavesa polymer matrix which contains a repeated “negative image” of thecompound and is able to selectively rebind the molecule under biologicalassay conditions (see, e.g., Ansell, et al., Current Opinion inBiotechnology (1996) 7:89-94 and in Shea, Trends in Polymer Science(1994) 2:166-173). Such “imprinted” affinity matrixes are amenable toligand-binding assays, whereby the immobilized monoclonal antibodycomponent is replaced by an appropriately imprinted matrix (see, e.g.,Vlatakis, et al., Nature (1993) 361:645-647). Through the use ofisotope-labeling, “free” concentration of compound can be readilymonitored and used in calculations of IC₅₀. Such “imprinted” affinitymatrixes can also be designed to include fluorescent groups whosephoton-emitting properties measurably change upon local and selectivebinding of compound. These changes can be readily assayed in real timeusing appropriate fiberoptic devices, in turn allowing the dose in atest subject to be quickly optimized based on its individual IC₅₀. Anexample of such a “biosensor” is discussed in Kriz, et al., AnalyticalChemistry (1995) 67:2142-2144.

Exemplary doses include milligram or microgram amounts of the compoundper kilogram of subject or sample weight, for example, about 1 microgramper kilogram to about 500 milligrams per kilogram, about 100 microgramsper kilogram to about 5 milligrams per kilogram, or about 1 microgramper kilogram to about 50 micrograms per kilogram. It is understood thatappropriate doses of a small molecule depend upon the potency of thesmall molecule with respect to the expression or activity to bemodulated. When one or more of these small molecules is to beadministered to an animal (e.g., a human) in order to modulateexpression or activity of a polypeptide or nucleic acid describedherein, a physician, veterinarian, or researcher may, for example,prescribe a relatively low dose at first, subsequently increasing thedose until an appropriate response is obtained. In addition, it isunderstood that the specific dose level for any particular animalsubject will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,gender, and diet of the subject, the time of administration, the routeof administration, the rate of excretion, any drug combination, and thedegree of expression or activity to be modulated.

The following examples are offered to illustrate but not to limit theinvention.

EXAMPLES

The following are exemplary procedures for synthesizing substitutedquinobenzoxazines analogs.

Example 1 Preparation of Substituted Quinobenzoxazine Analogs

The general synthetic scheme for the preparation of substitutedquinobenzoxazines analogs is shown in Scheme 1.

Ethyl-(2′,3′,4′,5′-tetrafluorobenzoyl)-ethanoate

Potassium ethyl malonate (3.66 g, 21.5 mmol), MgCl₂ (2.44 g, 25.7 mmol)and TEA (2.05 g, 20.3 mmol) were mixed in acetonitrile (70 ml) at 10-15°C for 2.5 hr. 2,3,4,5-tetrafluorobenzoyl chloride (2.00 g, 10.3 mmol) inacetonitrile (10 ml) was added at 0° C. over 15 min followed by a secondaddition of TEA (0.23 g, 2.3 mmol). After allowing to warm to RT, themixture was stirred for 16 hr. After removal of volatiles in vacuoToluene (30 ml) was added and removed in vacuo. Following the additionof toluene (60 ml), HCl 1.5 M (40 ml) was added cautiously, ensuring thetemperature did not exceed 25° C. The organic fraction was washed withHCl 1.5 M (2×25 ml) and water (2×25 ml), dried over MgSO₄ and reduced toa light orange oil in vacuo ([M+1]⁺ 265, 98%).

Ethyl-2-(2′,3′,4′,5′-tetrafluorobenzoyl-)-3-(dimethylamino)-prop-2-enoate

Dimethyl acetal dimethyl formamide (0.61 g, 5.1 mmol) was added dropwiseto ethyl-(2′,3′,4′,5′-tetrafluorobenzoyl)-ethanoate (0.9 g, 3.41 mmol)dissolved in acetic anhydride (2 ml), under argon. After 30 min solventwas removed in vacuo to leave the product as an orange oil in aquantitative yield ([M+1⁺] 320).

Ethyl1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-phenyl-phenoxazine-5-carboxylate

Ethyl-2-(2′,3′,4′,5′-tetrafluorobenzoyl-)-3-(dimethylamino)-prop-2-enoate(3.4 g, 8.0 mmol) and 2-amino-4-phenyl-phenol (1.5 g, 8.0 mmol) in 20DMSO (20 ml) was stirred under vacuum at 60° C. for 30 min. K2CO3 (5 g)and MeCN (20 ml) was added and the suspension was heated at 80° C. for 1hr. After cooling to RT, the mixture was poured into a slight excess ofdilute sulfuric acid and filtered. The product was recovered as ayellow-brown solid ([M+1]⁺ 420, 65%).

Example 2 Preparation of1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-phenyl-phenoxazine-5-carboxylicacid

Ethyl1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-phenyl-phenoxazine-5-carboxylate(2.2 g, 5.3 mmol) was refluxed in a mixture of conc. HCl and acetic acid(20 ml each) for 2 hr. After cooling to room temperature cold water (40ml) was added to the reaction mixture and the resulting precipitatefiltered and washed with ether to afford the product as a yellow-brownsolid 90% ([M+1]⁺ 392).

Example 3 Preparation ofEthyl-2-(2′,3′,4′,5′-tetrafluorobenzoyl-)-3-(napthyl-2″,3″-diamino)-prop-2-enoate

Ethyl-2-(2′,3′,4′,5′-tetrafluorobenzoyl-)-3-(dimethylamino)-prop-2-enoate(10.53 g, 33 mmol) in acetonitrile (50 ml) was added to a solution of2,3-diaminonapthalene (5.22 g, 33 mmol) in acetonitrile (150 ml),maintained at 50° C. under argon. After 3 hours, volatiles were removedin vacuo and the residue was subjected to chromatography over silica(15% EtOAc/Hexane) to yield the product as a yellow solid ([M+1]⁺ 433)(55%).

Example 4 Preparation of Ethyl1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]benzo[g]-phendiazine-5-carboxylate

Ethyl-2-(2′,3′,4′,5′-tetrafluorobenzoyl-)-3-(napthyl-2″,3″diamino)-prop-2-enoate(600 mg 1.4 mmol) was dissolved in a slurry of K₂CO₃ in DMF (500 ml),The mixture was stirred vigorously at 100° C. for 1 hour, then allowedto cool to RT. The K₂CO₃ was removed by filtration and the DMF removedin vacuo to leave a yellow-brown solid in quantitative yield. ([M+1]⁺393).

Example 5 Preparation of1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]benzo[g]-phendiazine-5-carboxylicacid

KOH solution (1N, 2.54 ml, 2.56 mmol) was added to a solution of ethyl1,2-difluoro-4-oxo-4H-pyrido[3,2,1-kl]benzo[g]-phendiazine-5-carboxylate(500 mg, 1.28 mmol) in ethanol (400 ml), heated under reflux. After 2hours the reaction mixture was allowed to cool to RT, then neutralizedwith HCl solution (1N). The product was collected by filtration as ayellow solid, 89%. ([M+1]+ 365).

Example 6 Preparation of Ethyl1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]-phenthiazine-5-carboxylateEthyl-2-(2′,3′,4′,5′-tetrafluorobenzoyl-)-3-(N-aminobenzyldisulfide)-prop-2-enoate

Ethyl-2-(2′,3′,4′,5′-tetrafluorobenzoyl-)-3-(dimethylamino)-prop-2-enoate(17.7 g, 55.3 mmol) in acetonitrile (10 ml) was added to a solution of1,2-aminothiophenol dimer (5.22 g, 33 mmol) in acetonitrile (100 ml).After 3 hours, volatiles were removed in vacuo and the residue wassubjected to chromatography over silica (1% MeOH/DCM) to yield theproduct as a yellow solid ([M+1]+ 523) (50%).

Ethyl 1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]-phenthiazine-5-carboxylate

Ethyl-2-(2′,3′,4′,5′-tetrafluorobenzoyl-)-3-(N-aminobenzyldisulfide)-prop-2-enoate(2.5 g 3.2 mmol) was dissolved in DMF (120 ml) and heated under refluxfor six hours. Removal of DMF in vacuo gave the product as a yellowsolid 90% ([M+1]+ 360).

Example 7 Preparation of1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]-phenthiazine-5-carboxylic acid

KOH solution (1N, 3.0, 3.0 mmol) was added to a solution of ethyl1,2-difluoro-4-oxo-4H-pyrido[3,2,1-kl]-phenthiazine-5-carboxylate (1000mg, 2.5 mmol) in ethanol (400 ml), heated under reflux. After 2 hoursthe reaction mixture was allowed to cool to RT, then neutralized withHCl solution (1N). The product was collected by filtration as a yellowsolid, ([M+1]+ 332, 95%)

Example 8 Preparation of5,6-Difluoro-9-hydroxy-3-oxo-3H-pyrido[3.2.1-kl]pyrimido[g]phenoxazine-2-carboxylicacid 7-nitroquinazoline-4,6-diol

To a solution containing 20 ml of 48% aqueous HBr and 20 ml of AcOH wasadded 6-methoxy-7-nitro-3,4-dihydroquinazolin-4-one (1.4 g, 6.3 mmol)and the mixture was refluxed overnight. The resulting solution wasevaporated to afford the crude phenol as a residue and was used withoutfurther purification (1.2 g, 5.8 mmol) (M+1, 208).

7-aminoquinazoline-4,6-diol

The crude product from above (1.0 g, 5.8 mmol) was diluted with 40 mlwater and 3 g of Tin II chloride dihydrate was added and the reactionwas stirred at room temperature. After 1 h the reaction was neutralizedwith K2CO3, and extracted with EtOAc (3×50 mL). The combined organicextracts were dried over sodium sulfate and the solvent was removed invacuo to afford the crude amino alcohol (1.0 g, 5.6 mmol) (M+1, 178).

Ethyl;5,6-difluoro-9-hydroxy-3-oxo-3H-pyrido[3,2,1-kl]pyrimido[g]phenoxazine-2-carboxylate

To a solution of the tetrafluoroenamine (2.2 g, 6.9 mmol) in DMSO (3 mL)was added the aminophenol (1.0 g, 5.6 mmol) and the reaction mixture wasstirred under vacuum (rotary evaporator) at 60° C. for 20 minutes. Thereaction mixture was allowed to cool to room temperature and was dilutedwith acetonitrile (200 mL) and potassium carbonate was added. Themixture was heated to reflux for 5 hours and poured into diluteHOAc/water. The solid product was collected by vacuum filtration anddried to afford the difluoroester as a tan solid (1.3 g, 3.2 mmol) (M+1,412).

5,6-Difluoro-9-hydroxy-3-oxo-3H-pyrido[3,2,1-kl]pyrimido[g]phenoxazine-2-carboxylicacid

The difluoroester (1.3 g, 3.2 mmol) was dissolved in a 1:1 mixture ofglacial acetic acid and 12 M HCl (20 mL) and refluxed for 30 min. Themixture was then cooled to room temperature and poured into water. Thesolid product was then collected by vacuum filtration and dried toafford the difluoroacid as a tan solid (0.98 g, 2.5 mmol) ([M+1]+ 392).

Example 9 Preparation of2-(2-(Ethoxycarbonyl)-5,6-difluoro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazin-9-yloxy)aceticacid Ethyl5,6-difluoro-9-hydroxy-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylate

To a solution of the tetrafluoroenamine (5.8 g, 18.2 mmol), dissolved inDMSO (12 mL), was added 2,4-dihydroxyaniline hydrochloride (2.5 g, 15.5mmol) and the mixture was heated to 60° C. under vacuum (rotaryevaporator) for 20 minutes. The reaction mixture was then diluted withacetonitrile (100 mL) and potassium carbonate (3 g) was added and themixture was refluxed overnight. The mixture was allowed to cool to roomtemperature and the solvent was removed in vacuo. A slight excess of 2 MHCl was added to rapidly dissolve the carbonate, and the solidprecipitate was filtered and dried to afford the difluoroester as a tansolid (5.0 g, 13.9 mmol) (M+1, 360).

2-(2-(Ethoxycarbonyl)-5,6-difluoro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazin-9-yloxy)aceticacid

To a solution of the difluoroester (2.1 g, 5.8 mmol) andtert-butylbromoacetate (2.0 g, 10.3 mmol) in DMF (30 mL) was addedpotassium carbonate (2.0 g) and the mixture was heated to 60° C. for 1hour. The reaction was allowed to cool and poured into water (500 mL)and extracted with ethyl acetate (3×100 mL),washed with brine, driedover magnesium sulfate and filtered over a pad of silica gel (30×50 mm),eluting with ethyl acetate. The solvent was removed in vacuo and theresulting material was triturated with hexanes and dried to afford thetert-butyl ester as a tan solid (2.8 g, 5.8 mmol). This material wasdissolved in trifluoroacetic acid (40 mL) and stirred at roomtemperature for 30 minutes. The solvent was removed in vacuo to affordthe acid as a tan solid (2.4 g, 5.7 mmol) (M+1, 418).

Example 10 Preparation of9-(Carboxymethoxy)-5,6-difluoro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylicacid

The difluoroester (2.4 g, 5.7 mmol) was dissolved in a 1:1 mixture ofglacial acetic acid and 12 M HCl (40 mL) and refluxed for 1 hour. Themixture was then cooled to room temperature and poured into water. Thesolid product was then collected by vacuum filtration and dried toafford the difluoroacid as a tan solid (2.0 g, 5.1 mmol) (M+1, 390).

Example 11 Preparation of Ethyl1,2,3-trifluoro-4-oxo-4H-pyrido[3,2,1-kl]benzo[g]-phenoxazine-5-carboxylate

To a solution of pentafluoroenamine (8 g, 23.7 mmol), prepared by asimilar procedure as for the tetrafluoroenamine dissolved in DMSO (12mL) was added 3-amino-2-naphthol (3.5 g, 21.9 mmol) and the mixture washeated to 60° C. under vacuum (rotary evaporator) for 2 hours. Thereaction mixture was then diluted with acetonitrile (200 mL) andpotassium carbonate (8.0 g) was added and the mixture was refluxedovernight. The mixture was allowed to cool to room temperature and thesolvent was removed in vacuo. A slight excess of 2 M HCl was added torapidly dissolve the carbonate, and the solid precipitate was filteredand dried to afford the difluoroester as a tan solid (1.3 g, 3.2 mmol)(M+1, 412).

Example 12 Preparation of1,2,3-Trifluoro-4-oxo-4H-pyrido[3,2,1-kl]benzo[g]-phenoxazine-5-carboxylicacid

The trifluoroester (1.3 g, 3.2 mmol) was dissolved in acetic acid (5 mL)and 12 M HCl was added (5 mL) and the reaction mixture was heated toreflux for 2 hours. The mixture was then cooled to room temperature,poured into water and the solid product was collected by vacuumfiltration and dried to afford the trifluoroacid as a pale solid (1.0 g,2.6 mmol) (M+1, 384).

Example 13 Preparation of Ethyl1,2-difluoro-4-oxo-4H-pyrido[3,2,1-kl]benzo[g]-phenoxazine-5-carboxylate

To a solution of the enamine (30 g, 94 mmol) in ethyl acetate (100 mL)was added 3-amino-2-naphthol (10 g, 63 mmol) at room temperature and themixture was immediately placed on a rotary evaporator and the solventwas removed over 2 hours at a temperature below 0° C. (ice formed on theflask) to produce a yellow solid. To this solid was added ether (200 mL)and the slurry was filtered to afford a yellow solid. This solid wasthen dissolved in DMF (200 mL) and potassium carbonate was added (16.5g, 120 mmol) and the mixture was heated to 90° C. for 1 hour. Themixture was allowed to cool to room temperature and water was added (500mL) and the resulting solid was filtered, washed with water and dried toafford the difluoroester as a tan solid (12.2 g, 30.8 mmol) (M+1, 394).

Example 14 Preparation of1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]benzo[g]-phenoxazine-5-carboxylicacid

The difluoroester (5 g, 12.7 mmol) was dissolved in methanol (50 mL) andconc HCl was added (20 mL) and the mixture was refluxed for 12 hours.The mixture was allowed to cool to room temperature and the solid wascollected by vacuum filtration, washing with methanol to afford thedifluoroacid as a light tan powder (3.6 g, 9.9 mmol) (M+1, 366).

Example 15 Preparation of Ethyl1-fluoro-4-oxo-4H-pyrido[3,2,1-kl]benzo[g]-phenoxazine-5-carboxylate

To a solution of the enamine, similarly prepared as thetetrafluoroenamine (14 g, 46.3 mmol) in ethyl acetate (100 mL) was added3-Amino-2-naphthol ( 5.0 g, 31.2 mmol) at room temperature and themixture was immediately placed on a rotary evaporator and the solventwas removed over 2 hours at a temperature below 0° C. (ice formed on theflask) to produce a yellow solid. To this solid was added methanol (200mL) and the slurry was filtered to afford a yellow solid. This solid wasthen dissolved in acetonitrile (200 mL) and potassium carbonate wasadded (10.0 g, 72.5 mmol) and the mixture was heated to 80° C. for 1hour. The mixture was allowed to cool to room temperature and water wasadded (500 mL) and the resulting solid was filtered, washed with waterand dried to afford the fluoroester as a tan solid (6.0 g, 16.0 mmol)(M+1, 376).

Example 16 Preparation of1-Fluoro-4-oxo-4H-pyrido[3,2,1-kl]benzo[g]-phenoxazine-5-carboxylic acid

The fluoroester (6.0 g, 16.0 mmol) was dissolved in acetic acid (10 mL)and 12 M HCl was added (10 mL) and the reaction mixture was heated toreflux for 2 hours. The mixture was then cooled to room temperature,poured into water and the solid product was collected by vacuumfiltration and dried to afford the fluoroacid as a pale solid (4.8 g,13.8 mmol) (M+1, 348).

Example 17 Preparation of Ethyl9-chloro-5,6-difluoro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylate

To a solution of the enamine (14.4 g, 45.3 mmol) in ethyl acetate (200mL) was added 5-chloro-2-aminophenol (5.0 g, 34.8 mmol) and the solventwas removed in vacuo with a rotary evaporator over 2 hours withoutheating. Methanol was added and the resulting phenolic enamine wasisolated by vacuum filtration. The resulting solid (7.0 g) was dissolvedin acetonitrile and potassium carbonate was added and the resultingmixture was heated to reflux for 2 hours. The mixture was then allowedto cool to room temperature and poured into Dilute HCl. The resultingsolid was collected by vacuum filtration and dried to afford thedifluoroester as a pale yellow solid (5.0 g, 13.3 mmol) (M+1, 378).

Example 18 Preparation of9-Chloro-5,6-difluoro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylicacid

The difluoroester (5.0 g, 13.3 mmol) was dissolved in acetic acid (45mL) and 12 M HCl was added (30 mL) and the reaction mixture was heatedto reflux for 2 hours. The mixture was then cooled to room temperature,poured into water and the solid product was collected by vacuumfiltration and dried to afford the difluoroacid as a pale solid (4.0 g,10.6 mmol) (M+1, 350).

Example 19 Preparation of Ethyl10-chloro-5,6-difluoro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylate

To a solution of the enamine (14.4 g, 45.3 mmol) in ethyl acetate (200mL) was added 4-chloro-2-aminophenol (5.0 g, 34.8 mmol) and the solventwas removed in vacuo with a rotary evaporator over 2 hours withoutheating. Methanol was added and the resulting phenolic enamine wasisolated by vacuum filtration. The resulting solid (7.5 g) was dissolvedin acetonitrile and potassium carbonate was added and the resultingmixture was heated to reflux for 2 hours. The mixture was then allowedto cool to room temperature and poured into Dilute HCl. The resultingsolid was collected by vacuum filtration and dried to afford thedifluoroester as a pale yellow solid (5.0 g, 13.3 mmol) (M+1, 378).

Example 20 Preparation of10-Chloro-5,6-difluoro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylicacid

The difluoroester (2.5 g) was dissolved in acetic acid (25 mL) and 12 MHCl was added (20 mL) and the reaction mixture was heated to reflux for2 hours. The mixture was then cooled to room temperature, poured intowater and the solid product was collected by vacuum filtration and driedto afford the difluoroacid as a pale solid (2.0 g, 5.3 mmol) (M+1, 350).

Example 21 Preparation of Ethyl5,6-Difluoro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylate

To a solution of the enamine (5.7 g, 17.9 mmol) in ethyl acetate (50 mL)was added 2-aminophenol (1.9 g, 17.43 mmol) at room temperature and themixture was immediately placed on a rotary evaporator and the solventwas removed over 2 hours at a temperature below 0° C. (ice formed on theflask) to produce a yellow solid. To this solid was added ether (25 mL)and the slurry was filtered to afford a yellow solid. This solid wasthen dissolved in DMF (20 mL) and potassium carbonate was added (2.9 g,21 mmol) and the mixture was heated to 90° C. for 1 hour. The mixturewas allowed to cool to room temperature and water was added (200 mL) andthe resulting solid was filtered, washed with water and dried to affordthe phenoxazine as a tan solid (2.9 g, 8.45 mmol) (M+1, 344).

Example 22 Preparation of5,6-Difluoro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylic acid

The difluoroester (5.0 g, 14 mmol) was dissolved in methanol (50 mL) andconc HCl was added (20 mL) and the mixture was refluxed for 2 hours. Themixture was allowed to cool to room temperature and the solid wascollected by vacuum filtration, washing with methanol to afford thedifluoroacid as a light tan powder (4.2 g, 13.3 mmol, 91%) (M+1, 316).

Example 23 Preparation of Ethyl1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]benzo[h]-phenoxazine-5-carboxylate

To a solution of the enamine (14.0 g, 45.3 mmol) in ethyl acetate (200mL) was added 1-amino-2-naphthol (5.0 g, 31.3 mmol) and the solvent wasremoved in vacuo with a rotary evaporator over 2 hours without heating.Methanol was added and the resulting phenolic enamine was isolated byvacuum filtration. The solid was dissolved in acetonitrile and potassiumcarbonate (10 g) was added and the mixture was heated to reflux for 2hours. The mixture was then allowed to cool to room temperature andpoured into Dilute HCl. The resulting solid was collected by vacuumfiltration and dried to afford the difluoroester as a pale yellow solid(5.0 g, 13.3 mmol) (M+1, 376).

Example 24 Preparation of1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]benzo[h]-phenoxazine-5-carboxylicacid

The difluoroester (5.5 g) was dissolved in acetic acid (25 mL) and 12 MHCl was added (20 mL) and the reaction mixture was heated to reflux for2 hours. The mixture was then cooled to room temperature, poured intowater and the solid product was collected by vacuum filtration and driedto afford the difluoroacid as a pale solid (5.0 g, 14.4 mmol) (M+1,348).

Example 25 Preparation of Ethyl1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]benzo[f]-phenoxazine-5-carboxylate

To a solution of the enamine (45 g, 141 mmol) in ethyl acetate (500 mL)was added 2-amino-1-naphthol (15.0 g, 93.8 mmol) and the solvent wasremoved in vacuo with a rotary evaporator over 2 hours without heating.Methanol was added and the phenolic enamine was isolated by vacuumfiltration. The resulting solid was dissolved in acetonitrile (400 mL)and potassium carbonate (25 g) was added and the mixture was heated toreflux for 2.5 hours. The mixture was then allowed to cool to roomtemperature and poured into Dilute HCl. The resulting solid wascollected by vacuum filtration and dried to afford the difluoroester asa pale yellow solid (19.69 g, 50.1 mmol) (M+1, 394).

Example 26 Preparation of1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]benzo[f]-phenoxazine-5-carboxylicacid

The difluoroester (15.0 g, 38.1 mmol) was dissolved in acetic acid (60mL) and 12M HCl was added (60 mL) and the reaction mixture was heated toreflux for 2 hours. The mixture was then cooled to room temperature,poured into water and the solid product was collected by vacuumfiltration and dried to afford the difluoroacid as a pale solid (11.7 g,32 mmol) (M+1, 366).

Example 27 Preparation of Ethyl1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]benzo[b]furan[2,3-g]-phenoxazine-5-carboxylate3-Aminodibenzofuran-2-ol

To a solution of the dibenzofuran (15 g, 70.4 mmol) dissolved inmethylene chloride (500 mL) at 0° C. was added BBr₃ (200 mL, 1 M inCH₂Cl₂) via addition funnel. After the addition was complete, themixture was allowed to come to room temperature over 1 hour and thenquenched with water followed by potassium carbonate (40 g). Theresulting solid was recovered by vacuum filtration and dried to affordthe hydroxyl dibenzofuran as a white solid (13.2 g, 199 mmol) (M+1,200).

Ethyl1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]benzo[b]furan[2,3-g]-phenoxazine-5-carboxylate

To a solution of the tetrafluoroenamine (15.0 g, 47 mmol) dissolved inDMSO (30 mL) was added the hydroxyl dibenzofuran (12.0 g, 60 mmol) andthe mixture was heated to 60° C. under vacuum (rotary evaporator) for 20minutes. The reaction mixture was then diluted with acetonitrile (200mL) and potassium carbonate (17 g) was added and the mixture wasrefluxed for 2.5 hours. The mixture was allowed to cool to roomtemperature and the solvent was removed in vacuo. A slight excess of 2 MHCl was added to rapidly dissolve the carbonate, and the solidprecipitate was filtered and dried to afford the difluoroester as a tansolid (15.0 g, 34.6 mmol) (M+1, 434).

Example 28 Preparation of1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]benzo[b]furan[2.3-g]-phenoxazine-5-carboxylicacid

The difluoroester (15.0 g, 34.6 mmol) was dissolved in acetic acid (60mL) and 12 M HCl was added (60 mL) and the reaction mixture was heatedto reflux for 2 hours. The mixture was then cooled to room temperature,poured into water and the solid product was collected by vacuumfiltration and dried to afford the difluoroacid as a pale solid (13.7 g,34 mmol) (M+1, 406).

Example 29 Preparation of Ethyl2-(ethoxycarbonyl)-5,6-difluoro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-10-carboxylate

To a solution of the tetrafluoroenamine (7.0 g, 21.9 mmol) dissolved inDMSO (25 mL) was added 4-hydroxy-3-amino benzoic acid (3.0 g, 19.6 mmol)and the mixture was heated to 60° C. under vacuum (rotary evaporator)for 2 hours. The reaction mixture was then diluted with acetonitrile(200 mL) and potassium carbonate (8.0 g) was added and the mixture wasrefluxed overnight. The mixture was allowed to cool to room temperatureand the solvent was removed in vacuo. A slight excess of 2 M HCl wasadded to rapidly dissolve the carbonate, and the solid precipitate wasfiltered and dried to afford the difluoroester as a tan solid (6.2 g,16.0 mmol) (M+1, 388).

Example 30 Preparation of2-(Ethoxycarbonyl)-5,6-difluoro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-10-carboxylicacid

The difluoroester (6.2, 16.0 mmol g) was dissolved in acetic acid (25mL) and 12 M HCl was added (20 mL) and the reaction mixture was heatedto reflux for 2 hours. The mixture was then cooled to room temperature,poured into water and the solid product was collected by vacuumfiltration and dried to afford the difluorodi-acid as a pale solid (5.3g, 14.8 mmol) (M+1, 360).

Example 31 Preparation of Ethyl5,6-difluoro-10-nitro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylate

A solution of the enamine (6.0 g, 18.7 mmol) and 2-amino-4-nitrophenol(3.5 g, 23.3 mmol) in acetonitrile was heated to 80° C. for 15 minutes.Potassium carbonate was then added (8.0 g) and the mixture was heated toreflux overnight. The reaction mixture was then filtered hot and thesolvent was removed in vacuo to afford the crude nitroester (5.0 g, 12.8mmol) (M+1, 389).

Example 32 Preparation of5,6-Difluoro-10-nitro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylicacid

The crude difluoroester (5.0 g, 12.8 mmol) was dissolved in acetic acid(25 mL) and 12 M HCl was added (20 mL) and the reaction mixture washeated to reflux for 2 hours. The mixture was then cooled to roomtemperature, poured into water and the solid product was collected byvacuum filtration and dried to afford the difluoroacid as a pale solid(2.0 g, 5.5 mmol) (M+1, 361).

Example 33 Preparation of Ethyl5,6-difluoro-3-oxo-10-phenyl-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylate

A solution of the enamine (5.4 g, 16.9 mmol) and3-amino-4-hydroxybiphenyl (3.5 g, 18.9 mmol) in acetonitrile was heatedto 80° C. for 90 minutes. Potassium carbonate was then added (8.0 g) andthe mixture was heated to reflux overnight. The reaction mixture wasthen filtered hot and the solvent was removed in vacuo to afford thecrude difluoroester (3.9 g, 9.3 mmol) (M+1, 420).

Example 34 Preparation of5,6-Difluoro-3-oxo-10-phenyl-3H-pyrido[3,2,1-kl]phenoxazine-2-carboxylicacid

The crude difluoroester (3.6 g, 8.6 mmol) was dissolved in acetic acid(10 mL) and 12 M HCl was added (10 mL) and the reaction mixture washeated to reflux for 2 hours. The mixture was then cooled to roomtemperature, poured into water and the solid product was collected byvacuum filtration and dried to afford the difluoroacid as a pale solid(2.6 g, 6.6 mmol) (M+1, 392).

Example 35 Preparation of Ethyl1,2-Difluoro-4-oxo-4H-11-sulfonic-acid-pyrido[3,2,1-kl]benzo[h]-phenoxazine-5-carboxylate

To a solution of the tetrafluoroenamine (5.4 g, 16.9 mmol) dissolved inDMSO (30 mL) was added 1-amino-2-hydroxy-4-naphthalenesulfonic acid (4.8g, 20 mmol) and the mixture was heated to 60° C. under vacuum (rotaryevaporator) for 2 hours. To the reaction mixture was added potassiumcarbonate (10.0 g) and the mixture was heated to 60° C. for 1 hour. Themixture was allowed to cool to room temperature and a slight excess of 2M HCl was added to rapidly dissolve the carbonate. The aqueous layer wasdecanted and the remaining organic residue was dissolved in methanol(100 mL) and precipitated with ethyl acetate (200 mL) and the solidprecipitate was filtered and dried to afford the sulfonic acid as abrown solid (3.1 g, 6.5 mmol) (M+1, 474).

Example 36 Preparation of1,2-Difluoro-4-oxo-4H-11-sulfonic-pyrido[3,2,1-kl]benzo[h]-phenoxazine-5-carboxylicacids

The crude difluoroester (1.5 g, 3.2 mmol) was dissolved in acetic acid(10 mL) and 12 M HCl was added (10 mL) and the reaction mixture washeated to reflux for 30 minutes. The solvent was removed in vacuo toafford the sulfonic acid as a brown solid (1.1 g, 2.5 mmol) (M+1, 446)

Example 37 Preparation of2-(Ethoxycarbonyl)-5,6-difluoro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-9-carboxylicacid

A solution of the difluoroenamine (5.2 g, 16.3 mmol) and4-amino-3-hydroxybenzoic acid (4.0 g, 26.1 mmol) in DMSO was stirred atroom temperature for 1.5 hours. Potassium carbonate (8 g) was then addedand the reaction mixture was stirred under vacuum (rotary evaporator)for 1 hour. The mixture was then heated to 100° C. for 1 hour and thenallowed to cool to room temperature. The reaction mixture was thenpoured into 1 M H₂SO₄ (500 mL) and the solids were recovered by vacuumfiltration. The resulting solid was dried to afford the crudedifluoroacid as a tan solid (5.0 g, 12.9 mmol) (M+1, 388).

Example 38 Preparation of5,6-Difluoro-3-oxo-3H-pyrido[3,2,1-kl]phenoxazine-2,9-dicarboxylic acid

The crude difluoroester (5.0 g, 12.9 mmol) was dissolved in acetic acid(20 mL) and 12 M HCl was added (20 mL) and the reaction mixture washeated to reflux for 1 hour. The reaction was allowed to cool to roomtemperature and water was added. The resulting solid was collected byvacuum filtration and dried overnight to afford the di-acid as a tansolid (1.9 g, 5.3 mmol) (M+1, 360).

Example 39 Preparation of Ethyl1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-fluorenone-5-carboxylate3-Nitro-2-hydroxyfluorenone

A solution of NO₂BF₄ (3.52 g, 25.5 mmol) in acetonitrile (100 ml) wasadded dropwise to a solution of 2-hydroxyfluorenone (5 g, 25.5 mmol) inacetonitrile (400 ml) at ambient temperature. The reaction mixture wasthen cooled to 0° C. and water (100 ml) was added to precipitateimpurities. After filtration, water (200 ml) was added and theprecipitate filtered off as a red solid (68%) (M+1, 242).

3-amino-2-hydroxyfluorenone

A mixture of 3-nitro-2-hydroxyfluorenone (1.6 g, 6.6 mmol) and SnCl₂ (3g, 6.6 mmol was refluxed in 100 ml acetic acid:conc. HCl (1:1) for 1hour. The mixture was allowed to cool to room temperature andneutralized with ammonium hydroxide. After extracting with EtOAc (3×100ml), combined organic fractions were dried over magnesium sulfate andevaporated to leave the product as a brown solid (65%) (M+1, 212).

Ethyl 1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-fluorenone-5-carboxylate

A mixture of 3-amino-2-hydroxyfluorenone (0.9 g, 4.26 mmol) andethyl-2-(2′,3′,4′,5′-tetrafluorobenzoyl-)-3-(dimethylamino)-prop-2-enoate(1.36 g, 4.26 m heated in DMSO (50 ml) under vacuum for 18 hr. Theproduct was extracted using EtOAc/Brine and the organic layers combinedand dried to give the product as a red solid. The solid was dissolved inDMSO (40 ml) containing a large excess of K₂CO₃ and heated at 100° C.for 30 min. After cooling to room temperature, brine (30 ml) was addedand the precipitated product collected as a yellow solid (60% over twosteps) (M+1, 446).

Example 40 Preparation of1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-fluorenone-5-carboxylic acid

A mixture of ethyl1,2-difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-fluorenone-5-carboxyl in aceticacid:conc. HCl (1:1) (50 ml each) was heated at reflux for 2 hr. Aftercooling to room temperature, water (50 ml) was added and the productcollected as yellow solid (94%) (M+1, 418).

Example 41 Preparation of Ethyl1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-anthraquinone-5-carboxylate

A mixture of 3-amino-2-hydroxyanthraquinone (5.54 g, 23.2 mmol) andethyl-2-(2′,3′,4′,5′-tetrafluorobenzoyl-)-3-(dimethylamino)-prop-2-enoate(8.7 g, 34.8 mm heated in a minimum of DMSO (˜10 ml) under vacuum for 24hr. The product was precipitated by the addition of water (50 ml). Thesolid was dried overnight in a vacuum oven and dissolved in DMSO (40 ml)containing a large excess of K₂CO₃ and heated at 100° C. for 30 min.After cooling to room temperature, brine (30 ml) was added and theprecipitated product collected as a yellow solid (60% over two steps)(M+1, 474).

Example 42 Preparation of1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-anthraquinone-5-carboxylic acid

A mixture of ethyl1,2-difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-anthraquinonenone-5-carboxylate(3.5 g, 6.8 mmol) in acetic acid:conc. HCl (1:1) (50 ml each) was heatedat reflux for 2 hr. After cooling to room temperature, water (50 ml) wasadded and the product collected as yellow solid (94%) (M+1, 446).

Example 43 Preparation of Ethyl1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-phenyl-phenoxazole-5-carboxylate2-amino(t-butoxy carbonyl)-5-amino hydroquinone

A solution of Boc anhydride (7.17 g, 33 mmol) and DIEA (17 ml, 99 mmol)in DMSO (20 ml) was added dropwise at room temperature to stirredsolution of 1,4-dihydroxy-2,5-diaminobenzene (7 g, 33 mmol). Afterstirring for 18 hr, the product was separated between EtOAc and brineand the organic layers combined and dried over MgSO₄. After removal ofsolvent the residue was subjected to column chromatography on silicaeluting with 25-50% EtOAc in hexane to give pure product (45%) (M+1,239).

4-hydroxy-3-amino(t-butoxy carbonyl)-phenoxazole

To solution of 2-amino(t-butoxy carbonyl)-5-amino hydroquinone (4.69 g,23.3 mmol) dissolved in acetonitrile/water (1:1; 20 ml) was added Nahydrosulfite (large excess) and the mixture stirred at room temp. for 15min. The acetonitrile was removed in vacuo and the aqueous mixtureextracted with EtOAc (3×20 ml). Combined organic layers were dried overMgSO₄ and solvent removed in vacuo. The residue was taken up in neattriethyl orthoformate (100 ml), left to stir for 16 hr then heated toreflux for 10 min. The product was precipitated following cooling toroom temp. by the addition of water (83%)(M+1, 251).

4-hydroxy-2-amino phenoxazole

4-Hydroxy-3-amino(t-butoxy carbonyl)-phenoxazole (3 g, 12 mmol) wasdissolved in neat TFA (100 ml) and allowed to stir at room temperaturefor 1 hour. TFA was removed in vacuo to leave the final product as a TFAsalt (quant.) (M−1, 149)

Ethyl-2-(2′,3′,4′,5′-tetrafluorobenzoyl-)-3-(N-(4-hydroxy-2-aminophenoxazole))-prop-2-enoate

A solution ofethyl-2-(2′,3′,4′,5′-tetrafluorobenzoyl-)-3-(dimethylamino)-prop-2-enoate(7.34 g, 23 mmol) and 2-amino-4-phenyl-phenol (3.45 g, 23 mmol) in EtOAc(20 ml) containing triethylamine (10 ml) was stirred under vacuum on therotary evaporator for 3 hours. The EtOAc was removed in vacuo and theresidue subjected to column chromatography on silica eluting with 50%EtOAc in hexane to give pure product (72%) (M+1, 425).

Ethyl1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-phenyl-phenoxazole-5-carboxylate

A solution ofethyl-2-(2′,3′,4′,5′-tetrafluorobenzoyl-)-3-(N-(4-hydroxy-2-aminophenoxazole))-prop-2-enoate (3.5 g, 8.25 mmol) in DMSO (50 m) containingK₂CO₃ (large excess) was heated at 80° C. for 10 min. After cooling toroom temperature, water was added to precipitate the product as a yellowsold (82%) (M+1, 385).

Example 44 Preparation of1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-(3′-hydroxy-4′-aminophenyl)-5-carboxylic acid

A mixture of ethyl1,2-difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-phenyl-phenoxazole-5-carboxylate(2.3 g, 6 mmol) in acetic acid:conc. HCl (1:1; 100 ml) was heated toreflux for 30 min. After cooling to room temp., volatiles were removedin vacuo to leave the product as a brown solid (82%) (M+1, 347).

Example 45 Preparation of1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-(nitro-phenoxazine)-5-carboxylicacid1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-(3′-hydroxy-4′-amino-(N-2″-fluoro-4″-nitrophenyl)-phenyl))-5-carboxylic acid

A solution of1,2-difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-(3′-hydroxy-4′-aminophenyl)-5-carboxylic acid (0.5 g, 1.44 mmol), 3,4-difluoro-nitro benzene(0.5 ml, 4.3 mmol) and DIEA (1 ml) was heated to 90° C. in NMP (50 ml)for 30 min. After cooling to room temp. the product was precipitated bythe addition of water and filtered (63%) (M+1, 486).

1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-(nitro-phenoxazine)-5-carboxylicacid

A solution of2-difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-(3′-hydroxy-4′-aminophenyl)-5-carboxylic acid (0.3 g, 0.6 mmol) in DMSO (50 ml) containingan excess of K₂CO₃ was stirred and heated to 110° C. for 1 hr. Aftercooling to room temp. the product was precipitated by the addition of 3MHCl and filtered (71%) (M+1, 465).

Example 46 Preparation of1,2-Difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-(amino-phenoxazine)-5-carboxylicacid

A mixture of1,2-difluoro-4-oxo-4H-pyrido[3,2,1-kl]-8-(nitro-phenoxazine)-5-carboxylacid (0.1 g, 0.2 mmol) and Tin (II) chloride (0.15 g, 0.6 mmol) inacetic acid:conc. HCl (1:1; 50 ml) was heated to reflux for four hr.After cooling to room temp. the product was precipitated by the additionof water and filtered (72%) (M+1, 435).

Example 47 Preparation of Preparation of amide derivatives ofsubstituted quinobenzoxazine analogs

To a series of solutions of the fluoroacid (0.5 mmol) in NMP (3.6 mL)was added the amines NHR₁R₂ (0.5-2.0 mmol) at room temperature. Thevessels were sealed and heated on a 90° C. hotplate with constantstirring for 1-2 hours until the reactions were determined to becomplete by HPLC/MS analysis. The reaction mixtures were allowed to coolto room temperature and water was added (20 mL). The resultingprecipitates were collected by vacuum filtration and dried under vacuum.In cases where 1.0 equivalent of amine was used, the resulting reactionmixtures were used in the next step “as is.” The resulting solids orsolutions were treated with HBTU (2.5 eq.) and DIEA in 3.6 mL NMP andallowed to stir for 30 minutes at room temperature under an inertatmosphere. These solutions were added to a series of amines NHR₃R₄ (2.5equivalents) in a 96 well format (Whatman Uniplate, 2 mL) and allowed toreact for 2 hours. Methanol was then added (50-100 μL) and the plate wasfiltered (Whatman Unifilter Polypropylene). The resulting liquids weredirectly chromatographed on reverse HPLC (Waters Xterra 19×50 mm) withmass directed collection (Micromass ZQ, Waters FCII). The fractions wereanalyzed for purity (MS TIC, UV) and dried by vacuum evaporation(Savant) with an average yield of 5-10 mg). Tables 1 and 2 listexemplary ester-substituted and amide-substituted quinobenzoxazinesanalogs, respectively.

Example 48 Antitumor Data

Two xenograft models for inoculation were harvested and diluted to aconcentration of 50×10⁶ cells/ml or 100×10⁶ cells/ml. Four to six weekold nude mice were injected with 0.1 ml of the cell suspension whichcontains between 5×10⁶ and 10×10⁶ cells. When tumors are of a suitablesize compound dosing is commenced. Tumor sizes are measured throughoutthe treatment period with calipers and body weights also measured.

Antitumor activities for compounds CX-3092 (1204) and CX-1535 (148) areshown in FIGS. 1 and 2, indicating efficacy (slow tumor weight gain) anda lack of toxicity (little body weight change). FIG. 1 shows theantitumor activity of compound 494 in Ramos, a model for fatal childhoodleukemia. FIG. 2 shows the antitumor activity of compound 516 inHCT-116, a model of colorectal cancer.

Example 49 Cell Proliferation and/or Cytotoxicity Assay

Cell culture. Human cervical epithelial cells (HeLa cells) were obtainedfrom American Type Culture Collection (Manassas, Va.). Cells were grownin Eagle's minimum essential medium (MEM, Hyclone, Utah) supplementedwith 2 mM Glutamine, 0.1 mM nonessential amino acid, 1 mM Na Pyruvate,1.5 g/L NaHCO₃, 50 mg/L gentamicin, and 10% fetal bovine serum (Hyclone,USA) in a humidified atmosphere of 5% CO₂ at 37° C.

MTS assays. Antiproliferative effects of anticancer drugs were tested bythe CellTiter 96 AQ_(ueous) assay (Promega, Wis.), which is acalorimetric assay for determining the number of viable cells. (See,e.g., Wang, L., et al., Methods Cell Sci (1996) 18:249-255).

Cells (4,500 cells/well) were seeded on 96 well flat bottom plates(Corning, N.Y.) in 100 μl of culture medium without any anticancer drugon day 0, and the culture medium was exchanged for that containedanticancer drugs at various concentrations on day 1. After incubationfor 3 days under normal growth conditions (on day 4), the monolayerswere washed once in PBS, and the medium was switched to 100 μl of PBS ineach of the 96 well plate. After mixing MTS and PMS at the ratio of20:1, 20 μl of MTS/PMS solution was added to each of the 96 well plateand incubated for 4 hours in a humidified atmosphere of 5% CO₂ at 37° C.The absorbance was read at 490 nm using FLUOstar Galaxy 96 well platereader (BMG Labtechnologies, Germany). μM concentrations (MTS data)reported in Tables 1-2 are concentrations at which 50% ofantiproliferative cell response is seen. Compounds whose IC₅₀ valueswere greater than 5 μM were not reported.

Example 50 Measurement of mRNA Values in Cell Assays

Real-time quantitative PCR (QPCR) method was used to detect accuratelythe changes of the target c-myc and the endogenous reference GAPDH genecopies in the same tube. Cells (15,000 cells/well) were seed on 96 wellflat bottom plates (Corning, N.Y.) and incubated under normal growthconditions for overnight. The next day, the culture medium was exchangedfor that contained anticancer drugs at various concentrations andincubate for 4 hrs in a humidified atmosphere of 5% CO₂ at 37° C. TotalRNA (tRNA) was extracted using the RNeasy 96 Kit (QIAGEN, CA). Theconcentration of the tRNA was determined by the RiboGreen RNAQuantitation Reagent (Molecular Probes, OR).

Reverse-transcription (RT) reaction was occurred using 50 ng of tRNAfrom each well in a 25 μl reaction containing 1× TaqMan RT buffer, 2.5uM random hexamers, 5.5 mM MgCl₂, 0.5 mM each deoxynucleosidetriphosphate (dNTP), 30 U MultiScribe Reverse Transcriptase, and 10 URNase inhibitor. RT reactions were incubated for 10 min at 25° C.,reverse-transcribed for 30 min at 48° C., inactivated for 5 min at 95°C., and placed at 4° C. All RT reagents were purchased from AppliedBiosystems, CA.

Real-Time QPCR reaction was performed in a 50 μl reaction containing the5 μl of cDNA, 1× Universal PCR Master Mix, 1×c-myc Pre-Developed Primersand Probe set, and 0.8×GAPDH Pre-Developed Primers and Probe set.Because of the relative abundance of GAPDH gene in Hela, GAPDH primersand probe concentration were adjusted to get accurate threshold cycles(C_(T)) for both genes in the same tube. The threshold cycle (C_(T))indicates the fractional cycle number at which the amount of amplifiedtarget reaches a fixed threshold. By doing so, the GAPDH amplificationwas stopped before it can limit the common reactants available foramplification of the c-myc, resulted in a reduction in ? Rn value ofGAPDH, but no effect on its C_(T) value, and equal amplificationefficiency for both genes. The ? Rn value represents the normalizedreporter signal minus the baseline signal. ? Rn increases during PCR asamplicon copy number increases until the reaction approaches a plateau.

The c-myc probe was labeled with 6FAM™ dye-MGB and the GAPDH probe waslabeled with VIC™ dye-MGB. Preincubation was performed for 2 min at 50°C. to activate AmpErase UNG enzyme and then for 10 min at 95° C. toactivate AmpliTaq DNA Polymerase. DNA was amplified for 40 cycles of 15sec at 95° C. and 1 min at 60° C. Human c-myc and GAPDH cDNA wereamplified, detected, and quantitated in real time using the ABI Prism7000 Sequence Detection system (Applied Biosystems, CA), which was setto detect both 6-FAM and VIC reporter dyes simultaneously.

The data was analyzed by using the ABI PRISM Sequence Detection Systemand Microsoft Excel. Relative quantitation was done using the standardcurve and comparative C_(T) method at the same time, and both methodsgave equivalent results. The cycle at which the amplification plotcrosses the C_(T) is known to accurately reflect relative mRNA values.(See, Heid, et al., Genome Res. (1996) 6:986-994; Gibson, et al., GenomeRes. (1996) 6:995-1001). QPCR reactions were set up in triplicate ateach cDNA sample and the triplicate C_(T) values were averaged. Allreagents including Pre-Developed Primers and probe set were purchasedfrom Applied Biosystems, CA. μM concentrations (STOP data) reported inTables 1-2 are concentrations at which 50% inhibition of c-myc mRNAlevels are seen. Compounds whose IC₅₀ values were greater than 5 μM werenot reported.

Example 51 Synthesis of CX-3092 and CX-3543

One method for synthesizing CX-3543 is shown below. As shown in Scheme2, CX-3543 is synthesized in a convergent manner, assembling thesubstructures 1, 1A and 2A in the final two synthetic steps (Scheme 2),to form CX-3543 having a 50:50 ratio of RS and SS isomers. CX-3092 maybe synthesized in a similar manner using a non-chiral form of 1A.

In more detail, pyrazinopyrrolidine 1A is synthesized via a [3+2]cycloaddition chemistry. Conversion of L-proline 7 tocyano-1-aminopyrrolidine 8 without loss of stereochemistry, followed byreduction provides the chiral 2-aminoethyl-1-methyl pyrrolidine 2A inhigh yield. CX-3543 was found to have a formulated solubility ofapproximately 20 mg/mL.

Example 52 In vitro Characterization for CX-3543

Various methods were used for in vitro characterization of the compoundsof the present invention, including but not limited to i) stop assays;ii) quadruplex/duplex selectivity screens; and iii) quadrome footprints.

Stop Assays. FIG. 3 represents a stop assay, a high throughput,first-pass screen for detecting drugs that bind to and stabilize thetarget G-quadruplex. As shown in FIG. 3, DNA template oligonucleotide iscreated, which contains the nucleotide sequence of the “target”quadruplex against which drug screening is desired. A fluorescentlylabeled primer DNA is then annealed to the 3′ end of the template DNA. ADNA polymerase such as Taq polymerase is then introduced to synthesize acomplementary strand of DNA by extending from the fluorescently labeledprimer. When the progress of the Taq polymerase is unhindered, itsynthesizes a full-length copy of the template. Addition of a test drugthat merely binds to duplex DNA but does not bind selectively thequadruplex region results in a decrease in synthesis of full lengthproduct and a concomitant increase in variable-length DNA copies. If,however, the test drug selectively binds to and stabilizes thequadruplex, the progress of polymerase arrests only at the quadruplex,and a characteristic “Stop Product” is synthesized.

In one aspect, the present invention provides compounds that willselectively target oncogenes regulated by the propeller/chair type ofquadruplex, such as c-myc. Compounds are initially screened at a singleconcentration, and “hits” are re-assayed over a range of doses todetermine an IC₅₀ value (i.e., the concentration of drug required toproduce an arrest product/full-length product ratio of 1:1). Theseproducts are visualized by capillary electrophoresis.

FIG. 4 shows the electrophoretogram for CX-3543 as well as for othertypes of drugs, including DNA interactive drugs such as the topo-IIpoison m-AMSA; the antibiotics distamycin, actinomycin D (a non-specifictranscription inhibitor), and mithramycin; and the antibioticfluoroquinolone ciprofloxacin. CX-3543 demonstrated an IC₅₀ value of 1.0μM in the stop assay, while none of the other DNA interactive drugsbound to the quadruplex at concentrations up to 100 μM. Thus, the firstpass screen effectively identified agents having at least a 100-foldselectivity for the propeller/chair type quadruplex.

Quadruplex/Duplex Selectivity Screens. The selectivity of CX-3543 forthe target quadruplex sequence relative to duplex DNA was measured usinga proprietary direct competition assay (i.e., “selectivity screen”).This selectivity screen uses the stop assay as a reporter system tomeasure the relative ability of an externally added DNA sequence tocompete with the target quadruplex structure formed in the DNA templatefor binding of the drug. In one example, the competitors are the c-mycquadruplex sequence, which is identical to the quadruplex sequencepresent in the template DNA; or a plasmid DNA which mimics complexgenomic duplex DNA. The degree to which each competitor successfully“soaks up” drug in solution is reflected by the quantitative decrease insynthesis of the stop product. In this manner, the relative bindingaffinities of drug to both the target quadruplex and duplex DNA aredetermined.

FIG. 5 shows the quadruplex/duplex binding selectivity of CX-3543. Asshown in FIG. 5, the free c-myc quadruplex (myc27) competes moreeffectively for the drug than does the plasmid DNA. The ratio of thecompetitor IC₅₀ values indicates relative binding affinities of the drugfor the quadruplex or duplex DNA. For CX-3543, this selectivity ratiofor myc-quadruplex/plasmid DNA is around 400.

Quadrome Footprints. CX-3543 was also evaluated for its ability to bindto other native quadruplex structures of biological relevance, includingquadruplex control elements that regulate a range of differentoncogenes. The resulting data are used to create the Quadrome footprint.

FIG. 6 shows the Quadrome footprint for CX-3543. In FIG. 6, the primaryIC₅₀ data (upper panel) derived from different quadruplexes are invertedand displayed in column chart format to identify affected oncogenes(second panel). This chart reveals that CX-3543 selectively binds tothose quadruplexes that are capable of adopting the chair/propellerconformation (see, boxed oncogene cluster in Quadrome).

In vitro range of antitumor activity. The antitumor range of action ofCX-3543 was tested in vitro using a panel of cancer cell-lines. Cellswere exposed to CX3543 at various concentrations over a four day period,and IC₅₀ values were calculated (Table 4). In particular examples, IC₅₀values (i.e., concentrations of test drug required to induce 50% celldeath) were measured by Alamar Blue. As shown in Table 4, CX-3543demonstrates a broad range of antitumor action. TABLE 4 MDA- MDA- CellHCT- HT- MB- MB- Line A549 116 HeLa 29 231 468 MiaPaca PC3 Ramos HepG2786-O IC₅₀ 2.9 3.3 3.3 5.5 2.4 1.5 2.8 3.2 0.3 2.5 2.1 (μM)

Furthermore, CX-3543 was evaluated for potential inhibitory activityagainst topoisomerase I and topoisomerase II enzymes, and was determinednot to be an inhibitor or poison of either enzyme. FIG. 7 illustratesdata from a toposiomerase-II poison assay, using VP-16 (Etoposide) as acontrol Topoisomerase-II poison. In the presence of 1 μM VP-16,topoisomerase-II mediates the formation of double-strand breaks withinthe supercoiled DNA, giving rise to linear DNA product. CX-3543 did notinduce topoisomerase dependent strand breaks at concentrations as highas 100 μM.

Example 53 Drug Metabolism and Pharmacokinetics of CX-3543

CX-3543 was evaluated for i) potential inhibitory activity againstcytochrome P450 isoenzymes; and ii) metabolic stability with humanhepatocytes. The pharmacokinetics of CX-3543 was also evaluated.

Cytochrome P450 (CYP450) Inhibition Assay. CX-3543 was tested in an invitro inhibition assay with five CYP450 isoenzymes: CYP1A2, CYP2C9,CYP2C19, CYP2D6 and CYP3A4. No significant inhibition was apparent withfour of the five isoenzymes: CYP1A2, CYP2C9, CYP2C19 and CYP2D6. In thecase of the remaining isoenzyme, CYP3A4, only minor and partialinhibition in the 1-3 μM range was evident. These results suggest thatat clinical levels of drug exposure, there is a low likelihood ofdrug-drug interactions with the liver metabolism of co-administereddrugs. It should not, therefore, be necessary to adjust the doses ofco-administered anticancer cancer drugs that are principally metabolizedby the CYP450 isoenzyme system (e.g., docetaxel).

Metabolic Stability Assay and Metabolite Profiling. The metabolicstability of CX-3543 with human hepatocytes was evaluated followingincubation at 0, 30, 60, and 120 minutes at 1 and 20 μM. Consumption ofthe parent compound and the appearance of metabolites were carefullymonitored, and mass spectrometer product ion scans were conducted onpotential metabolite peaks to define the metabolite fragmentationpattern. The consumption of CX-3543 was noted to be moderate or slow,and no major metabolite peaks from hepatic biotransformation wereobserved. These results suggest that hepatic clearance of CX-3543 willbe moderate to low, and the minor role of hepatic elimination minimizesthe likelihood of drug-drug interactions with co-administered compoundsthat are inducers or inhibitors of liver enzymes.

Pharmacokinetic Study. A pharmacokinetic study to determine the timecourse of CX-3543 disposition was conducted in mice followingintravenous administered of 25 mg/kg of CX-3543. Regular sampling wasundertaken throughout a 24 hour period after IV dose administration, andthe key derived pharmacokinetic parameters were derived. FIG. 8 providesthe graphical representation of the pharmacokinetic profile of CX-3543from which key parameters (Table 5) were obtained.

In FIG. 8, C_(max) is the maximum CX-3543 plasma concentration attained(expressed in ng/ml); AUC_(0-inf) is the area under the CX-3543 plasmaconcentration versus time curve, extrapolated from time zero to infinity(expressed in ng.hr/ml); T_(1/2) is the terminal half life of CX-3543(expressed in hr); Vd_(ss) is the volume of distribution of CX-3543 atsteady state (expressed in L/kg); and Cl_(S) is the systemic clearanceof CX-3543 (expressed in L/hr/kg). As shown in FIG. 8, CX-3543 has aC_(max) of about 5353 ng/mL; an AUC_(0-inf) of about 20332.5 ng.hr/ml;and a T_(1/2) of about 4.6 hr. TABLE 5 PARAMETER VALUE UNITS C_(max)4353.0 ng/mL AUC_(0-inf) 20322.5 ng · hr/mL T_(1/2) 4.6 hr Vd_(ss) 7.26L/kg Cl_(S) 1.23 L/hr/kg

Example 54 In vivo Evaluation in Xenograft Models

CX-3543 was tested against a number of cancer xenograft models todetermine the in vivo efficacy. In-house testing for activity in nudemice was first conducted using the HCT-116 colorectal xenograft model,followed by the PC3 prostate cancer xenograft model in which both c-mycand VEGF oncogenic proteins are highly over-expressed. As shown in FIG.9, CX-3543 inhibits tumor growth in HCT-116 colorectal cancer xenograftmodel. As shown in FIG. 10, CX-3543 inhibits tumor growth in PC3prostate cancer xenograft model. Thus, representative tumor volumegraphs from both tumor models show suppression of cancer growth whentumor bearing mice are administered intraperitoneal doses of CX-3453.

It is understood that the foregoing detailed description andaccompanying examples are merely illustrative, and are not to be takenas limitations upon the scope of the invention. Various changes andmodifications to the disclosed embodiments will be apparent to thoseskilled in the art. Such changes and modifications, including withoutlimitation those relating to the chemical structures, substituents,derivatives, intermediates, syntheses, formulations and/or methods ofuse of the invention, may be made without departing from the spirit andscope thereof. U.S. patents and publications referenced herein areincorporated by reference. TABLE 1 Stop Data MTS Data Structure c-Myc μMHella μM 1

4 2

2.5 3

2.5 4

1.76 5

1.75 7.20 6

1.75 7

1.75 8

1.75 9

1.75 10

1.75 11

1.75 12

1.75 13

1.75 14

1.75 15

1.75 16

0.9 17

0.75 18

0.75 19

0.75 20

0.75 21

0.75 22

0.75 23

0.75 24

0.5 7.00 25

0.25 0.20

TABLE 2 26

4 0.73 27

3 3.80 28

3 2.50 29

3 2.00 30

3 1.80 31

3 1.40 32

3 0.60 33

3 0.29 34

3 0.28 35

3 0.21 36

3 0.16 37

2.5 2.80 38

2.5 0.26 39

2.5 40

2.5 41

2.5 42

2.5 43

2.5 44

2.5 45

2.5 46

2.5 47

2.5 48

2.5 49

2.5 50

2.5 51

2.5 52

2.5 53

2.5 54

2.5 55

2.5 56

2.5 57

2.5 58

2.5 59

2.5 60

2.5 61

2.5 62

2.5 63

2.5 64

2.5 65

2.5 66

2.5 67

2.5 68

2.5 69

2.5 70

2.5 71

2.5 72

2.5 73

2.5 74

2.5 75

2.5 76

2.5 77

2.5 78

2.5 79

2.5 80

2.5 81

2.5 82

2.5 83

2.5 84

2.5 85

2.5 86

2.5 87

2.5 88

2.5 89

2.5 90

2.5 91

2.5 92

2.5 93

2.5 94

2.5 95

2.5 96

2.5 97

2.5 98

2.5 99

2.5 100

2.5 101

2.25 102

1.8 2.20 103

1.8 104

1.75 2.80 105

1.75 2.80 106

1.75 2.50 107

1.75 1.80 108

1.75 0.46 109

1.75 0.31 110

1.75 0.25 111

1.75 0.22 112

1.75 0.22 113

1.75 114

1.75 115

1.75 116

1.75 117

1.75 118

1.75 119

1.75 120

1.75 121

1.75 122

1.75 123

1.75 124

1.75 125

1.75 126

1.75 127

1.75 128

1.75 129

1.75 130

1.75 131

1.75 132

1.75 133

1.75 134

1.75 135

1.75 136

1.75 137

1.75 138

1.75 139

1.75 140

1.75 141

1.75 142

1.75 143

1.75 144

1.75 145

1.75 146

1.75 147

1.75 148

1.75 149

1.75 150

1.75

Stop Data MTS Data Structure c-Myc μM Hella μM 151

1.75 152

1.75 153

1.75 154

1.75 155

1.75 156

1.75 157

1.75 158

1.75 159

1.75 160

1.75 161

1.75 162

1.75 163

1.75 164

1.75 165

1.75 166

1.75 167

1.75 168

1.75 169

1.75 170

1.75 171

1.75 172

1.75 173

1.75 174

1.75 175

1.75 176

1.75 177

1.75 178

1.75 179

1.75 180

1.75 181

1.75 182

1.75 183

1.75 184

1.75 185

1.75 186

1.75 187

1.75 188

1.75 189

1.75 190

1.75 191

1.75 192

1.75 193

1.75 194

1.75 195

1.75 196

1.75 197

1.75 198

1.75 199

1.75 200

1.75 201

1.75 202

1.75 203

1.75 204

1.75 205

1.75 206

1.75 207

1.75 208

1.75 209

1.75 210

1.75 211

1.75 212

1.75 213

1.75 214

1.75 215

1.75 216

1.75 217

1.75 218

1.75 219

1.75 220

1.75 221

1.75 222

1.75 223

1.75 224

1.75 225

1.75 226

1.75 227

1.75 228

1.75 229

1.75 230

1.75 231

1.75 232

1.75 233

1.75 234

1.75 235

1.75 236

1.75 237

1.75 238

1.75 239

1.75 240

1.75 241

1.75 242

1.75 243

1.75 244

1.75 245

1.75 246

1.75 247

1.75 248

1.75 249

1.75 250

1.75 251

1.75 252

1.75 253

1.75 254

1.75 255

1.75 256

1.75 257

1.75 258

1.75 259

1.75 260

1.75 261

1.75 262

1.75 263

1.75 264

1.75 265

1.75 266

1.75 267

1.75 268

1.75 269

1.75 270

1.75 271

1.75 272

1.75 273

1.75 274

1.75 275

1.75 276

1.75 277

1.75 278

1.75 279

1.75 280

1.75 281

1.75 282

1.75 283

1.75 284

1.75 285

1.75 286

1.75 287

1.75 288

1.75 289

1.75 290

1.75 291

1.75 292

1.75 293

1.75 294

1.75 295

1.75 296

1.75 297

1.75 298

1.75 299

1.75 300

1.75

Stop Data MTS Data Structure c-Myc μM Hella μM 301

1.75 302

1.75 303

1.5 2.10 304

1.13 305

1.05 306

1 3.20 307

1 3.10 308

Chiral 1 3.10 309

1 3.00 310

1 2.30 311

Chiral 1 2.10 312

1 1.90 313

1 314

1 315

1 316

1 317

1 318

1 319

Chiral 1 320

1 321

1 322

Chiral 1 323

1 324

1 325

1 326

1 327

1 328

1 329

1 330

1 331

1 332

1 333

1 334

1 335

1 336

1 337

1 338

1 339

Chiral 0.94 340

0.9 8.50 341

Chiral 0.9 0.28 342

0.9 343

0.9 344

0.9 345

0.9 346

0.9 347

0.9 348

0.9 349

0.9 350

0.9 351

0.9 352

0.9 353

0.9 354

0.9 355

Chiral 0.89 356

Chiral 0.85 357

Chiral 0.75 8.60 358

Chiral 0.75 5.70 359

0.75 4.80 360

Chiral 0.75 4.50 361

0.75 4.20 362

0.75 4.00 363

0.75 3.80 364

0.75 3.80 365

0.75 3.80 366

0.75 3.70 367

0.75 3.70 368

0.75 3.60 369

0.75 3.50 370

0.75 3.50 371

Chiral 0.75 3.50 372

0.75 3.40 373

0.75 3.30 374

0.75 3.30 375

0.75 2.70 376

Chiral 0.75 2.40 377

0.75 2.20 378

0.75 2.10 379

0.75 1.90 380

0.75 1.80 381

0.75 1.80 382

Chiral 0.75 1.80 383

0.75 0.37 384

0.75 0.37 385

0.75 0.36 386

0.75 0.34 387

0.75 0.33 388

0.75 0.31 389

0.75 0.29 390

0.75 0.24 391

0.75 0.24 392

0.75 0.19 393

0.75 394

0.75 395

0.75 396

0.75 397

0.75 398

0.75 399

0.75 400

0.75 401

0.75 402

Chiral 0.75 403

Chiral 0.75 404

Chiral 0.75 405

Chiral 0.75 406

0.75 407

0.75 408

0.75 409

Chiral 0.75 410

Chiral 0.75 411

Chiral 0.75 412

Chiral 0.75 413

Chiral 0.75 414

Chiral 0.75 415

0.75 416

Chiral 0.75 417

0.75 418

0.75 419

0.75 420

0.75 421

Chiral 0.75 422

0.75 423

0.75 424

0.75 425

0.75 426

0.75 427

Chiral 0.75 428

Chiral 0.75 429

Chiral 0.75 430

0.75 431

0.75 432

0.75 433

0.75 434

0.75 435

0.75 436

0.75 437

0.75 438

0.75 439

0.75 440

0.75 441

0.75 442

0.75 443

0.75 444

Chiral 0.75 445

Chiral 0.75 446

0.75 447

0.75 448

0.75 449

0.75 450

0.75 451

0.75 452

0.75 453

0.75 454

0.75 455

0.75 456

0.75 457

0.75 458

Chiral 0.75 459

0.75 460

0.75 461

0.75 462

0.75 463

0.75 464

0.75 465

0.75 466

0.75 467

0.75 468

Chiral 0.74 469

Chiral 0.73 470

0.64 471

0.64 472

0.62 3.30 473

0.62 474

Chiral 0.58 0.37 475

Chiral 0.58 0.24 476

0.58 477

0.55 2.10 478

0.53 479

0.5 7.40 480

Chiral 0.5 3.70

Stop Data MTS Data Structure c-Myc μM Hella μM 481

0.5 3.60 482

0.5 3.40 483

0.5 3.20 484

0.5 3.10 485

0.5 0.50 486

0.5 0.39 487

0.5 0.18 488

0.5 489

0.5 490

0.5 491

0.45 492

0.45 493

0.44 0.40 494

0.44 0.19 495

0.42 496

0.41 4.00 497

0.41 2.10 498

0.41 499

0.4 500

0.375 5.60 501

0.375 4.20 502

0.375 4.00 503

0.375 4.00 504

0.375 3.40 505

0.375 3.40 506

0.375 3.40 507

0.375 3.30 508

0.375 3.20 509

0.375 3.10 510

0.375 3.10 511

0.375 3.10 512

0.375 3.10 513

0.375 3.10 514

0.375 2.90 515

0.375 2.50 516

0.375 2.30 517

0.375 2.20 518

0.375 2.20 519

0.375 2.10 520

0.375 1.90 521

0.375 1.70 522

0.375 1.70 523

0.375 1.60 524

0.375 1.50 525

0.375 1.,2 526

0.375 0.90 527

0.375 0.79 528

0.375 0.75 529

0.375 0.75 530

0.375 0.72 531

0.375 0.48 532

0.375 0.44 533

0.375 0.40 534

0.375 0.40 535

0.375 0.31 536

0.375 0.31 537

0.375 0.29 538

0.375 0.28 539

0.375 0.28 540

0.375 0.27 541

0.375 0.27 542

0.375 0.23 543

0.375 0.20 544

0.375 0.20 545

0.375 0.15 546

0.375 0.10 547

0.375 0.10 548

0.375 0.10 549

0.375 550

0.375 551

0.375 552

0.375 553

0.375 554

0.375 555

0.375 556

0.375 557

0.375 558

0.375 559

0.375 560

0.37 561

0.34 562

0.32 0.85 563

0.25 0.31 564

0.25 0.29 565

0.25 0.20 566

0.25 0.03 567

0.25 568

0.22 4.10 569

0.18 7.80 570

0.18 6.80 571

0.18 4.80 572

0.18 4.80 573

0.18 4.50 574

0.18 4.00 575

0.18 2.10 576

0.18 2.10 577

0.18 1.10 578

0.18 0.58 579

0.18 0.49 580

0.18 0.30 581

0.18 0.30 582

0.18 0.28 583

0.18 0.25 584

0.18 0.19 585

0.18 586

0.18 587

0.18 588

0.18 589

0.18 590

0.18 591

0.13 3.30 592

0.1 3.80 1467

0.25 0.35 1468

0.375 0.35 1469

1.75 1470

1.75 1471

5

TABLE 3 Stop Structure Data e-mye μM MTS Data Helia μM 1472

1473

1474

1475

1476

593

594

595

596

597

598

599

600

601

602

603

604

605

606

607

608

609

610

611

612

613

614

615

616

617

618

619

620

621

622

623

624

625

626

627

628

629

630

631

632

633

634

635

636

637

638

639

640

641

642

643

644

645

646

647

648

649

650

651

652

653

654

655

656

657

658

659

660

661

662

663

664

665

666

667

668

669

670

671

672

673

674

675

676

677

678

679

680

681

682

683

684

685

686

687

688

689

690

691

692

693

694

695

696

697

698

699

700

701

702

703

704

705

706

707

708

709

710

711

712

713

714

715

716

717

718

719

720

721

722

723

724

725

726

727

728

729

730

731

732

733

734

735

736

737

Stop Data MTS Data Structure c-Myc μM Hella μM 738

739

740

741

742

743

744

745

746

747

748

749

750

751

752

753

754

755

756

757

758

759

760

761

762

763

764

765

766

767

768

769

770

771

772

773

774

775

776

777

778

779

780

781

782

783

784

785

786

787

788

789

790

791

792

793

794

795

796

797

798

799

800

801

802

803

804

805

806

807

808

809

810

811

812

813

814

815

816

817

818

819

820

821

822

823

824

825

826

827

828

829

830

831

832

833

834

835

836

837

838

839

840

841

842

843

844

845

846

847

848

849

850

851

852

853

854

855

856

857

858

859

860

861

862

863

864

865

866

867

868

869

870

871

872

873

874

875

876

877

878

879

880

881

882

883

884

885

886

887

888

889

890

891

Stop Data MTS Data Structure c-Myc μM Hella μM 892

893

894

895

896

897

898

899

900

901

902

903

904

905

906

907

908

909

910

911

912

913

914

915

916

917

918

919

920

921

922

923

924

925

926

927

928

929

930

931

932

933

934

935

936

937

938

939

940

941

942

943

944

945

946

947

948

949

950

951

952

953

954

955

956

957

958

959

Chiral 960

961

962

963

964

965

966

967

968

969

970

971

972

973

974

975

976

977

978

979

980

981

982

983

984

985

986

987

988

989

990

991

992

993

994

995

996

997

998

Chiral 999

1000

1001

1002

1003

1004

1005

1006

1007

1008

1009

1010

1011

1012

1013

1014

1015

1016

1017

1018

1019

1020

1021

1022

1023

1024

1025

1026

1027

1028

1029

1030

1031

1032

1033

1034

1035

1036

1037

1038

1039

1040

1041

Stop Data MTS Data Structure c-Myc μM Hella μM 1042

1043

1044

1045

1046

1047

1048

1049

1050

1051

1052

1053

1054

1055

1056

1057

1058

1059

1060

1061

1062

1063

1064

1065

1066

1067

1068

1069

1070

1071

1072

1073

1074

1075

1076

1077

1078

1079

1080

1081

1082

1083

1084

1085

1086

1087

1088

1089

1090

1091

1092

1093

1094

1095

1096

1097

1098

1099

1100

1101

1102

1103

1104

1105

1106

1107

1108

1109

1110

1111

1112

1113

1114

1115

1116

1117

1118

1119

1120

1121

1122

1123

1124

1125

1126

1127

1128

1129

1130

1131

1132

1133

1134

1135

1136

1137

1138

1139

1140

1141

1142

1143

1144

1145

1146

1147

1148

1149

1150

1151

1152

1153

1154

1155

1156

1157

1158

1159

1160

1161

1162

1163

1164

1165

1166

1167

1168

1169

1170

1171

1172

1173

1174

1175

1176

1177

1178

1179

1180

1181

1182

1183

1184

1185

1186

1187

1188

1189

1190

1191

Stop Data MTS Data Structure c-Myc λM Hella λM 1192

1193

1194

1195

1196

1197

1198

1199

1200

1201

1202

1203

1204

1205

1206

1207

1208

1209

1210

1211

1212

1213

1214

1215

1216

1217

1218

1219

1220

1221

1222

1223

1224

1225

1226

1227

1228

1229

1230

1231

1232

1233

1234

1235

1236

1237

1238

1239

1240

1241

1242

1243

1244

1245

1246

1247

1248

1249

1250

1251

1252

1253

1254

1255

1256

1257

1258

1259

1260

1261

1262

1263

1264

1265

1266

1267

1268

1269

1270

1271

1272

1273

1274

1275

1276

1277

1278

1279

1280

1281

1282

1283

1284

1285

1286

1287

1288

1289

1290

1291

1292

1293

1294

1295

1296

1297

1298

1299

1300

1301

1302

1303

1304

1305

1306

1307

1308

1309

1310

1311

1312

1313

1314

1315

1316

1317

1318

1319

1320

1321

1322

1323

1324

1325

1326

1327

1328

1329

1330

1331

1332

1333

1334

1335

1336

1337

1338

1339

1340

1341

Stop Data MTS Data Structure c-Myc λM Hella αM 1342

1343

1344

1345

1346

1347

1348

1349

1350

1351

1352

1353

1354

1355

1356

1357

1358

1359

1360

1361

1362

1363

1364

1365

1366

1367

1368

1369

1370

1371

1372

1373

1374

1375

1376

1377

1378

1379

1380

1381

1382

1383

1384

1385

1386

1387

1388

1389

1390

1391

1392

1393

1394

1395

1396

1397

1398

1399

1400

1401

1402

1403

1404

1405

1406

1407

1408

1409

1410

1411

1412

1413

1414

1415

1416

1417

1418

1419

1420

1421

1422

1423

1424

1425

1426

1427

1428

1429

1430

1431

1432

1433

1434

1435

1436

1437

1438

1439

1440

1441

1442

1443

1444

1445

1446

1447

1448

1449

1450

1451

1452

1453

1454

1455

1456

1457

1458

1459

1460

1461

1462

1463

1464

1465

1466

1. A compound having formula (1A),

and pharmaceutically acceptable salts, esters and prodrugs thereof. 2.The compound of claim 1, wherein said compound is chiral.
 3. Apharmaceutical composition comprising the compound of claim 1 and apharmaceutically acceptable excipient.
 4. The pharmaceutical compositionof claim 3, comprising 2% w/w of the compound of claim
 1. 5. Thepharmaceutical composition of claim 4, further comprising 4% w/wmannitol.
 6. The pharmaceutical composition of claim 4, furthercomprising 0.5% sucrose.
 7. The pharmaceutical composition of claim 3,wherein said formulation is aqueous.
 8. The pharmaceutical compositionof 3, wherein said formulation has a pH of about 3.5:
 9. Thepharmaceutical composition of claim 3, wherein said formulation issuitable for injection, oral, parenteral, intravenous, intramuscular,topical or subtopical administration.
 10. The pharmaceutical compositionof claim 3, comprising 2% w/w of the compound of claim 1, 4% w/wmannitol, and 0.5% sucrose.
 11. The pharmaceutical composition of claim10, wherein said composition is aqueous.
 12. The pharmaceuticalcomposition of claim 10, wherein said formulation has a pH of about 3.5.13. The pharmaceutical composition of claim 10, wherein said formulationis suitable for injection.
 14. A method for ameliorating a cellproliferative disorder, comprising administering to a subject in needthereof an effective amount of the compound of claim 1 or apharmaceutical composition thereof, thereby ameliorating saidcell-proliferative disorder.
 15. The method of claim 14, wherein saidcell proliferative disorder is cancer.
 16. The method of claim 14,wherein cell proliferation is reduced, or cell death is induced.
 17. Themethod of claim 14, wherein said subject is human or an animal.
 18. Amethod for reducing cell proliferation or inducing cell death,comprising contacting a system with an effective amount of the compoundof claim 1 or a pharmaceutical composition thereof, thereby reducingcell proliferation or inducing cell death in said system.
 19. The methodof claim 18, wherein said system is a cell or tissue.
 20. A method forreducing microbial titers, comprising contacting a system with aneffective amount of the compound of claim 1 or a pharmaceuticalcomposition thereof, thereby reducing microbial titers.
 21. The methodof claim 20, where the system is a cell or tissue.
 22. The method ofclaim 20, wherein the microbial titers are viral, bacterial or fungaltiters.
 23. A method for ameliorating a microbial infection, comprisingadministering to a subject in need thereof an effective amount of thecompound of claim 1 or a pharmaceutical composition thereof, therebyameliorating said microbial infection.
 24. The method of claim 23, wherethe subject is a human or an animal.
 25. The method of claim 23, whereinsaid microbial infection is viral, bacterial or fungal.
 26. Thepharmaceutical composition of claim 3, further comprising achemotherapeutic agent.
 27. The pharmaceutical composition of claim 10,further comprising a chemotherapeutic agent.
 28. The method of claim 14,further comprising administering a chemotherapeutic agent.
 29. Themethod of claim 18, further comprising administering a chemotherapeuticagent.